JP2021533180A - Methods and compositions for modifying plants - Google Patents

Abstract

As used herein, a plant modification composition comprising a plurality of plant messenger packs (PMPs), including, for example, plant extracellular vesicles (EV) or fragments, portions or extracts thereof, wherein the PMP is a plant. Disclosed is a plant-modifying composition comprising a modifier, wherein the plant-modifying agent comprises heterologous RNA. The compositions herein are useful in methods for modifying plants, such as to increase fitness for plants, where increased fitness for plants is resistant to development, growth, yield, and abiotic stressors. Or it includes increased resistance to biological stressors. [Selection diagram] None

Description

Sequence Listing This application is submitted electronically in ASCII format and includes a sequence listing incorporated herein by reference in its entirety. The ASCII copy made on August 23, 2019 is named 51296-004WO3_Sequence_Listing_08.23.19_ST25 and is 12,136 bytes in size.

In agriculture, the delivery of bioactive agents to plants to induce the desired plant modification can be hampered in a number of ways. For example, plant modifiers such as nucleic acids or peptides may be sensitive to degradation during delivery. In addition, plant modifiers need to infiltrate various plant parts, tissues, vascular systems and / or cells in order to modify the plant. Therefore, there is a need for methods and compositions for effectively delivering plant modifiers to plants in the art.

The present specification discloses a plant modification composition containing a plurality of plant messenger packs (PMPs) carrying a plant modifier. The present composition is useful in a method of delivering a plant modifying agent by a method of introducing a desired plant trait, such as increased fitness of the plant.

In a first aspect, herein is a method of administering heterologous RNA to a plant comprising delivering a composition comprising a plurality of plant messenger packs (PMPs) to the plant, each of the plurality being heterologous. A method is provided in which the composition comprises RNA and is delivered at an effective concentration for increasing the adaptability of the plant.

In some embodiments, the heterologous RNA is a guide RNA (gRNA). In some embodiments, the gRNA is a component of a ribonucleoprotein complex (RNP), each of the plurality of PMPs comprising an RNP.

In some embodiments, the PMP is a lipid rearrangement PMP (LPMP).

In some embodiments, the heterologous RNA is encapsulated by PMP.

In some embodiments, each of the PMPs comprises purified plant extracellular vesicles (EVs).

In some embodiments, PMP in the composition is an effective concentration for modifying plant traits, the modification increasing the fitness of the plant, eg development, growth, yield, abiotic stressor. Increases resistance to or resistance to biological stressors. In some embodiments, an increase in plant adaptability is a modification of flowering time, and an increase in plant adaptability is an improvement in the quality of the product harvested from the plant, the taste of the product harvested from the plant, Improving appearance or shelf life or reducing the production of allergens that stimulate the immune response in animals.

In another aspect, as used herein, a plant modification composition comprising a plurality of PMPs, wherein the PMP comprises a plant modification agent, is provided. In some embodiments, PMP in the composition is a concentration effective for modifying plant traits such as plant fitness. In some embodiments, PMP in the composition is a concentration effective for increasing the fitness of the plant.

In another embodiment, herein, a plant modification composition comprising a plurality of plant messenger packs (PMPs), the PMP comprising a plant modifier, and the PMP in the composition altering plant traits. A plant modification composition is provided that is an effective concentration for the modification and increases the adaptability of the plant.

In some embodiments of the compositions described herein, an increase in fitness is an increase in development, proliferation, yield, resistance to abiotic stressors or resistance to biological stressors. In some embodiments, increased plant adaptability is disease resistance, drought resistance, heat resistance, cold resistance, salt resistance, metal resistance, herbicide resistance, drug resistance, water utilization efficiency, nitrogen utilization, nitrogen stress tolerance. , Nitrogen fixation, pest resistance, herbicide resistance, pathogen resistance, yield, yield under water shortage conditions, vitality, growth, photosynthetic ability, nutrition, protein content, carbohydrate content, oil content, biomass, sprout length, root length, Increases in root structure, fruit set, seed weight, fruit set or amount of harvestable product. In some embodiments, the increased fitness is early flowering. In some embodiments, increasing fitness of a plant is an improvement in the quality of the product harvested from the plant. In some embodiments, increasing fitness of a plant is an improvement in the taste, appearance or shelf life of the product harvested from the plant. In some embodiments, the increased fitness is a decrease in the production of allergens that stimulate the immune response in the animal.

In some embodiments of the compositions described herein, the plant modifier is a heterologous nucleic acid.

In another embodiment, herein, a plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP comprises a heterologous nucleic acid, and the PMP in the composition is for modifying a plant. A plant modification composition is provided that is an effective concentration and the modification increases the adaptability of the plant.

In another embodiment, herein, a plant modification composition comprising a plurality of plant messenger packs (PMPs), the PMP comprising a heterologous nucleic acid, and the PMP in the composition increases the adaptability of the plant. A plant modification composition is provided, which is an effective concentration for the production.

In some embodiments where the plant modifier is a heterologous nucleic acid, the heterologous nucleic acid is a DNA, RNA, PNA or hybrid DNA-RNA molecule. In some embodiments, the RNA is messenger RNA (mRNA), guide RNA (gRNA) or inhibitory RNA. In some embodiments, the inhibitory RNA is RNAi, shRNA or miRNA. In some embodiments, inhibitory RNA inhibits gene expression in plants.

In some embodiments where the plant modifier is a heterologous nucleic acid, the nucleic acid is an enzyme, pore-forming protein, signaling ligand, cell membrane permeabilizing peptide, transcription factor, acceptor, antibody, nanobody, gene editing protein, in the plant. An mRNA, modified mRNA or DNA molecule that increases the expression of a riboprotein, protein aptamer or chaperon. In some embodiments, increased expression in plants is about 5%, 10%, 15%, 20%, 30%, 40%, 50%, relative to reference levels (eg, expression in untreated plants). 60%, 70%, 80%, 90%, 100% or more than 100% increased expression. In some embodiments, increased expression in plants is about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold relative to reference levels (eg, expression in untreated plants). The expression is increased by a factor of 5, about 50, about 75 times, or about 100 times or more.

In some embodiments, the nucleic acid is an antisense RNA that reduces the expression of enzymes, transcription factors, secretory proteins, structural factors, riboproteins, protein aptamers, chapelons, receptors, signaling ligands or transport factors in plants. , SiRNA, shRNA, miRNA, aiRNA, PNA, morpholino, LNA, piRNA, ribozyme, DNAzyme, aptamer, cyclRNA, gRNA or DNA molecule. In some embodiments, the reduction in expression in plants is about 5%, 10%, 15%, 20%, 30%, 40%, 50%, relative to reference levels (eg, expression in untreated plants). Expression reduction of 60%, 70%, 80%, 90%, 100% or more than 100%. In some embodiments, the reduction in expression in plants is about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold relative to reference levels (eg, expression in untreated plants). The expression is reduced by a factor of 5, about 50, about 75 times, or about 100 times or more.

In some embodiments where the plant modifier is a heterologous nucleic acid, the heterologous nucleic acid is a construct that is not integrated into the plant genome. In other embodiments, the heterologous nucleic acid is a construct that integrates (eg, stably integrates) into the plant genome.

In some embodiments of the compositions described herein, the plant modifier is a heterologous peptide. In some embodiments, the peptide is an enzyme, pore-forming protein, signaling ligand, cell membrane penetrating peptide, transcription factor, receptor, antibody, Nanobody, gene editing protein, riboprotein, protein aptamer or chaperone. In some embodiments, the peptide reduces gene expression in a plant. In some embodiments, the peptide increases gene expression in a plant.

In yet another embodiment, in the present specification, a plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP comprises a heterologous peptide, and the PMP in the composition is a plant adaptability. A plant modification composition is provided that is an effective concentration for increasing. In some embodiments, the peptide is an enzyme, pore-forming protein, signaling ligand, cell membrane penetrating peptide, transcription factor, receptor, antibody, Nanobody, gene editing protein, riboprotein, protein aptamer or chaperone. In some embodiments, the peptide reduces gene expression in a plant. In some embodiments, the peptide increases gene expression in a plant.

In some embodiments of the compositions described herein, the PMP varies from two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Contains various plant modifiers. In some embodiments, the two or more different plant modifiers include heterologous nucleic acids and heterologous peptides. In certain embodiments, the heterologous plant modifier is one listed in the section entitled "Heterologous Plant Modifier" in the detailed description herein.

In some embodiments of the compositions described herein, the plant modifier is encapsulated by PMP.

In some embodiments of the compositions described herein, the plant modifier is embedded in the surface of the PMP.

In some embodiments of the compositions described herein, the plant modifier is conjugated to the surface of the PMP.

In some embodiments, the composition is at least 24 hours (eg, at least 24 hours, 30 hours or 40 hours), at least 48 hours (eg, at least 48 hours (= 2 days)), 3 days, 4 days, 5 days. Days or 6 days), at least 7 days (eg, at least 7 days (= 1 week), at least 2 weeks, at least 3 weeks or at least 4 weeks) or at least 30 days (eg, at least 30 days, at least 60 days or at least 90) (Days) Stable. In some embodiments, the composition is at least 24 ° C. (eg, at least 24 ° C., 25 ° C., 26 ° C., 27 ° C., 28 ° C., 29 ° C., 30 ° C., 37 ° C., 42 ° C. or> 42 ° C.). At least 20 ° C (eg, at least 20 ° C, 21 ° C, 22 ° C or 23 ° C), at least 4 ° C (eg, at least 5 ° C, 10 ° C or 15 ° C), at least −20 ° C (eg, at least −20 ° C, − At a temperature of 15 ° C, -10 ° C, -5 ° C or 0 ° C) or at least -80 ° C (eg, at least -80 ° C, -70 ° C, -60 ° C, -50 ° C, -40 ° C or -30 ° C). stable. In some embodiments, PMP is stable in liquid nitrogen (about -195.8 ° C.). In some embodiments, the composition is stable at room temperature for at least 1 day and / or at 4 ° C. for at least 1 week. In some embodiments, the composition is stable under UV light. In some embodiments, the composition is stable over the period specified herein at temperatures in the natural habitat of the plant.

In some embodiments of the compositions described herein, PMP comprises a plurality of proteins (ie, PMP proteins), the concentration of PMP can be measured as the concentration of PMP protein therein. In some embodiments, the plurality of PMPs in the composition is at least 0.025 μg / ml PMP protein (eg, at least 0.025, 0.05, 0.1 or 0.5 μg / ml PMP protein). , At least 1 μg / ml PMP protein (eg, at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 μg / ml PMP protein), at least 10 μg / ml PMP protein (eg, at least 10, 15). , 20, 25, 30, 35, 40 or 45 μg / ml PMP protein), at least 50 μg / ml PMP protein (eg, at least 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 μg / ml PMP protein), at least 100 μg / ml PMP protein (eg, at least 100, 125, 150, 175, 200 or 225 μg / ml PMP protein), at least 250 μg / ml PMP protein (eg, at least 250, 300, The concentration of 350, 400, 450 or 500 μg / ml PMP protein) or at least 500 μg / ml PMP protein (eg, at least 500, 600, 700, 800 or 900 μg / ml PMP protein). In some embodiments, the plurality of PMPs in the composition is at least 1 mg / ml of PMP protein (eg, at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 PMP proteins / ml). ) Or at least 10 mg / ml of PMP protein (eg, at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg / ml PMP protein).

In some embodiments of the compositions described herein, PMP comprises purified plant extracellular vesicles (EVs) or fragments or extracts thereof. In some embodiments, the plant EV is a modified plant extracellular vesicle (EV). In certain embodiments, the plant EV is a plant exosome or plant microvesicles. In some embodiments, the PMP comprises a plant EV marker such as that outlined in the Appendix.

In some embodiments of the compositions described herein, the plurality of PMPs can be pure. For example, the composition may be substantially free of plant organelles such as chloroplasts, mitochondria or nuclei (eg, having less than 25%, 20%, 15%, 10%, 5%, 2%). be.

In some embodiments of the compositions described herein, the plant is an agricultural plant or a horticultural plant. In some embodiments, the agricultural plant is a soybean plant, a wheat plant, a corn plant, a tomato plant or an alfalfa plant.

In some embodiments of the compositions described herein, the composition is formulated for delivery to a plant. In some embodiments, the composition is formulated for delivery to a plant leaf, pollen, seed, root, fruit, sprout or flower. In some embodiments, the composition comprises an agriculturally acceptable carrier.

In some embodiments of the compositions described herein, the composition is formulated to stabilize PMP.

In some embodiments of the compositions described herein, the plant modification composition is non-insecticidal.

In some embodiments of the compositions described herein, the composition is formulated as a liquid, solid, aerosol, paste, gel or gas composition.

In another aspect, herein is a plant modification composition comprising a plurality of PMPs, wherein the PMP is (a) a step of providing an initial sample from the plant or a portion thereof, wherein the plant or a portion thereof. Steps Containing EV; (b) Isolating the crude PMP fraction from the initial sample, wherein the crude PMP fraction is from the plant or a portion thereof at a reduced level relative to the level in the initial sample. A step comprising at least one contaminant or unwanted component of; (c) Purifying a crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs are crude EVs. A step of loading a plant modifier into multiple pure PMPs, having at least one contaminant or unwanted component from the plant or portion thereof, at a reduced level relative to the level in the fraction. ; And an plant modification composition produced by a process comprising (e) optionally formulating the PMP of step (d) for delivery to the plant is provided. In yet another embodiment, the present specification provides a plant comprising any of the plant modification compositions provided herein.

In yet another embodiment, herein is a method of delivering a plant modification composition to a plant, the step of contacting the plant with any composition of the plant modification composition provided herein. Methods to include are provided.

In yet another embodiment, the present specification comprises a method of modifying a plant, comprising delivering to the plant an effective amount of any of the plant modification compositions provided herein. Modifications thereby introduce beneficial traits in the plant or against untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, Methods of increasing 60%, 70%, 80%, 90%, 100% or more than 100%) are provided.

In yet another embodiment, the present specification comprises a method of increasing the fitness of a plant, comprising delivering to the plant an effective amount of any of the plant modification compositions provided herein. , Fitness of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90) %, 100% or more than 100%) methods are provided.

In some embodiments of the methods described herein, the plant modification composition is delivered to leaves, seeds, pollen, roots, fruits, sprouts, flowers or portions thereof. In some embodiments, the plant modification composition is delivered to plant cells. In some embodiments, the plant modification composition is delivered to a hypoplastic protoplast. In some embodiments, the plant modification composition is delivered to the tissue of the plant. For example, the composition can be delivered to a plant meristem (eg, apical meristem, lateral meristem or internode meristem). In some examples, the composition may be delivered to plant permanent tissue (eg, single tissue (eg, parenchyma, collenchyma or thick tissue) or complex permanent tissue (eg, xylem or phloem)). .. In some examples, the composition is delivered to the plant germ.

In yet another embodiment, a method of increasing fitness of a plant, wherein the pollen of the plant is contacted with an effective amount of any of the plant modification compositions provided herein. Adaptation of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%). , 90%, 100% or more than 100%).

In yet another embodiment, herein, a method of increasing the fitness of a plant, the step of contacting the seeds of a plant with an effective amount of any of the plant modification compositions provided herein. Adaptation of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%). , 90%, 100% or more than 100%).

In yet another embodiment, the present specification comprises contacting a plant protoplast with an effective amount of any of the plant modification compositions provided herein, comprising adapting the plant to an untreated plant. Increased degree (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or over 100%) A way to make it is provided.

In yet another embodiment, herein, a method of increasing the fitness of a plant, the plant cells of the plant are contacted with an effective amount of any of the plant modification compositions provided herein. Plant fitness to untreated plants, including steps (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80). %, 90%, 100% or more than 100%) methods are provided.

In yet another embodiment, herein, a method of increasing the fitness of a plant, the meristem of the plant is contacted with an effective amount of any of the plant modification compositions provided herein. Plant fitness to untreated plants, including steps (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80). %, 90%, 100% or more than 100%) methods are provided.

In yet another embodiment, herein, a method of increasing the fitness of a plant, the step of contacting the plant germ with an effective amount of any of the plant modification compositions provided herein. Adaptation of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%). , 90%, 100% or more than 100%).

In some embodiments of the methods described herein, increased fitness is in development, growth, yield, resistance to abiotic stressors or resistance to biological stressors (eg, about 1%, 2%,). 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) increase.

In some embodiments of the methods described herein, increased plant adaptability is disease resistance, drought resistance, heat resistance, cold resistance, salt resistance, metal resistance, herbicide resistance, drug resistance, water utilization. Efficiency, nitrogen utilization, nitrogen stress resistance, nitrogen fixation, pest resistance, herbicide resistance, pathogen resistance, yield, yield under water shortage conditions, vitality, growth, photosynthetic capacity, nutrition, protein content, carbohydrate content, oil content, In the amount of biomass, shoot length, root length, root structure, fruit set, seed weight, fruit set or harvestable product (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40). %, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) increase. In some embodiments, the increased fitness is early flowering. In some embodiments of the methods described herein, the increase in fitness is in the quality of the product harvested from the plant (eg, about 1%, 2%, 5%, 10%, 20%, 30). %, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) improvement.

In some embodiments of the methods described herein, increasing fitness of a plant is an improvement in the taste or appearance of the product harvested from the plant.

In some embodiments of the methods described herein, the increase in fitness of the plant is during the storage period of the product harvested from the plant (eg, about 1%, 2%, 5%, 10%, 20). %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) increase.

In some embodiments of the methods described herein, increased fitness is (eg, about 1%, 2) in the production of allergens (eg, pollen) that stimulate an immune response in animals (eg, humans). %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) reduction.

In some embodiments of the methods herein, the plant is an agricultural plant or a horticultural plant. In some embodiments, the plant is a soybean plant, a wheat plant, a corn plant, a tomato plant or an alfalfa plant.

In some embodiments of the methods herein, the plant modification composition is delivered as a liquid, solid, aerosol, paste, gel or gas.

In yet another embodiment, provided herein is an agricultural formulation comprising any of the compositions described herein and a carrier or excipient suitable for agricultural use. The pharmaceutical product can be in liquid, solid (eg, granules, pellets, powder, dry fluid or wet powder), aerosol, paste, gel or gaseous form. The pharmaceutical product is configured to be diluted (eg, the composition is a soluble or water-dispersible solid), sprayed, applied, injected or applied to a plant, soil or seed. Or it can be combined using the instruction manual).

In yet another embodiment, the specification provides a kit comprising an instruction manual for use as any of the compositions described herein and as a plant modification composition. In some embodiments of any of the above embodiments, the PMP is a lipid rearrangement PMP (LPMP). In some embodiments, the LPMP comprises a foreign cationic lipid. In some embodiments, each of the modified PMPs is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90%. Contains cationic lipids of. In some embodiments, the cationic lipid is DOTAP or DC-cholesterol.

Other features and advantages of the invention will become apparent from the detailed description and claims.

Definitions As used herein, the term "plant-modifying composition" is a composition comprising a plurality of PMPs, wherein the PMP relates to a composition comprising a plant-modifying agent.

As used herein, "delivery" or "contact" is either directly on the plant or adjacent to the plant in areas where the composition is effective for altering the fitness of the plant. Refers to applying a plant modification composition to a plant. In a method in which the composition is in direct contact with the plant, the composition may be in contact with the entire plant or only a portion of the plant.

As used herein, the terms "effective amount", "effective concentration" or "effective concentration for" modify a plant in or on a target plant (eg, increase the adaptability of the plant). Refers to the amount of PMP or heterologous plant modifier in it sufficient to achieve the above results to reach the target level (eg, defined level or threshold level).

As used herein, the term "formulated for delivery to a plant" refers to a plant modification composition comprising an agriculturally acceptable carrier. As used herein, the term "agriculturally acceptable" carrier or excipient is, for example, a carrier or excipient suitable for use in agriculture, for use on plants. .. In certain embodiments, the agriculturally acceptable carrier or excipient is for a human or animal consuming the resulting produce that is commensurate with a reasonable benefit / risk ratio from the plant, environment or derived from them. Has no adverse side effects that are not appropriate.

As used herein, "increasing plant adaptability" refers to an increase in plant adaptability that results directly as a direct result of contact with the plant modification compositions described herein, eg. Improved yield, improved plant vitality or improved quality or quantity of products harvested from the plant, improved pre-harvest or post-harvest traits that may be desirable for agriculture or gardening (eg, taste, appearance, storage). Includes improvement of traits (eg, reduced allergen production) that are beneficial to humans in terms of duration) or otherwise. The improved yield of the plant is compared to the yield of the same product of the plant produced under the same conditions or compared to the application of conventional plant modifiers (eg, plant modifiers delivered without PMP). However, without applying this composition, the product of the plant in measurable amounts (eg, as measured by plant biomass, cereals, seed or fruit yield, protein content, carbohydrate or oil content or leaf area). Regarding the increase in yield. For example, the yields are at least about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50. %, About 60%, about 70%, about 80%, about 90%, about 100% or more than 100% can be increased. Yields can be expressed in terms of the amount of weight or volume of the plant or plant product based on some criteria. Criteria can be expressed in terms of time, area of growth, weight of plant produced or amount of raw material used. Increased adaptation of plants also includes other means, such as increased or improved assessment of vitality, increased planting (number of plants per unit area), increased plant height, increased stem perimeter, plant canopy. Increased appearance, improved appearance (visually measured dark green leaf color), improved root evaluation, increased seedling development, protein content, increased leaf size, increased number of leaves, less dead roots Increased leaf emergence, sprout strength, reduced nutrient or fertilizer requirements, increased seed germination, increased young shoot productivity, earlier flowering, earlier grain or seed maturation, less plant lodging (verse) ( Induction), increased sprout growth, or any combination of these factors, in measurable or significant amounts exceeding the same factor in plants produced under the same conditions, but without administration of the composition or conventional. It can also be measured by administering an agricultural action agent of.

As used herein, the term "heterogeneous" (1) (eg, originates from a plant or non-plant part from which PMP is produced) (eg, using the loading approach described herein). It is exogenous to the plant (added to the PMP) or (2) endogenous to the plant cell or tissue from which the PMP is produced, but higher than naturally found (eg, naturally occurring). Either was present in the PMP at a concentration (higher than that found in plant extracellular vesicles) (eg, added to the PMP using the loading approach, genetic engineering, in vitro or in vivo approach described herein). Refers to a drug (eg, a plant modifier).

As used herein, the term "functional agent" is PMP or is associated with PMP using in vivo or in vitro methods (eg, loaded into or on PMP (eg, encapsulated by PMP). Drugs (eg, modifying plants) that can be (eg, embedded in PMP or conjugated to PMP)) and produce the listed results according to the compositions and methods. Peptide or nucleic acid).

As used herein, the terms "nucleic acid" and "polynucleotide" are replaceable and length (eg, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20). , 30, 40, 50, 100, 150, 200, 250, 500, 1000 or more nucleic acids), linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. Point to. The term also includes RNA / DNA hybrids. Nucleic acids are typically linked in nucleic acids by phosphate diester binding, but the term "nucleic acid" refers to other types of binding or skeleton (eg, among others, phosphoroamide, phosphorothioate, phosphorodithioate, O-methyl). Also included are nucleic acid analogs with phosphoroamidat, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA) and peptide nucleic acid (PNA) binding or skeleton). The nucleic acid can be single-stranded, double-stranded, or contain both moieties of the single-stranded and double-stranded sequences. Nucleic acids include any combination of deoxyribonucleotides and ribonucleotides and, for example, adenine, thymine, cytosine, guanine, uracil and modified or non-standard bases (eg, hypoxanthin, xanthin, 7-methylguanine, 5,6-dihydrouracil, 5). -Can include any combination of bases, including (including methylcytosine and 5-hydroxymethylcytosine cytosine).

As used herein, the term "peptide", "protein" or "polypeptide" is used in length (eg, at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 16). 18, 20, 25, 30, 40, 50, 100 amino acids or more), with or without post-translational modifications (eg, glycosylation or phosphorylation) or, for example, one or more non-aminoacyls covalently linked to a peptide. Includes naturally occurring or non-naturally occurring amino acid chains (either D-amino acids or L-amino acids), regardless of the presence of groups (eg, sugars, lipids, etc.), eg, natural proteins, synthetic or recombinant. Polypeptides and peptides, hybrid molecules, peptoids or peptide mimetics of.

As used herein, "percent identity" between two sequences is referred to as Altschul et al. , (1990) J.M. Mol. Biol. 215: Determined by the BLAST 2.0 algorithm described in 403-410. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds and their progeny. Plant cells include, without limitation, seeds, suspension cultures, embryos, meristem regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen or microspore cells. Plant sites include, but are not limited to, roots, stems, shoots, leaves, pollen, seeds, fruits, harvests, tumor tissues, sap (eg, xylem juice and phloem juice) and various forms of cells and cultures. Includes differentiated and undifferentiated tissues, including objects (eg, single cells, protoplasts, embryos and callus tissues). The plant tissue can be that of a plant or that of a plant organ, tissue or cell culture. In addition, the plant can be genetically engineered, for example, to produce a heterologous protein or RNA of any of the plant modification compositions in the methods or compositions described herein.

As used herein, the terms "plant extracellular vesicle", "plant EV" or "EV" refer to a naturally occurring encapsulated lipid bilayer structure in a plant. Optionally, the plant EV comprises one or more plant EV markers. As used herein, the term "plant EV marker" refers to, but is not limited to, components naturally associated with plants such as plant proteins, protein nucleic acids, plant small molecules, plant lipids or combinations thereof. Includes any of the plant EV markers listed in. In some examples, the plant EV marker is a discriminating marker for the plant EV marker, but not an agricultural agent. In some examples, the plant EV marker is a discriminating marker for the plant EV marker and is also an agricultural agent (eg, bound to or encapsulated in multiple PMPs, or with multiple PMPs. Not directly bound or enclosed in them).

As used herein, the term "plant messenger pack" or "PMP" has a diameter of about 5 to 2000 nm (eg, at least 5 to 1000 nm, at least 5 to 500 nm, at least 400 to 500 nm, at least 25 to 250 nm, Refers to lipid structures of at least 50-150 nm or at least 70-120 nm (eg, lipid double layer, monolayer, multi-layer structure; eg, follicular lipid structure), which are lipid or non-lipid components bound to them (eg, eg. , Peptide, nucleic acid or small molecule), obtained from plant origin or fragments, portions or extracts thereof (eg, enriched, isolated or purified), concentrated, isolated or purified from plants, plant parts or plant cells. In this case, concentration or isolation is the removal of one or more contaminants or unwanted components from the plant of origin. PMP can be a highly purified preparation of naturally occurring EV. Preferably, contaminants or unwanted components from the plant of origin, one or more contaminants or unwanted components from the plant of origin, such as plant cell wall components; pectin; plant organella (eg, mitochondria; chloroplasts, leucoplasts). Or at least pigments such as amyloplasts; and nuclei; plant chromatin (eg, plant chromosomes); or plant molecular aggregates (eg, protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates or lipid-protein structures). 1% (eg at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90% , 95%, 96%, 98%, 99% or 100%) are removed. Preferably, the PMP is for one or more contaminants or unwanted components from the plant of origin when measured by weight (w / w), spectral imaging (% transmittance) or conductivity (S / m). At least 30% pure (eg, at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure or at least 100% pure). ..

In some examples, the PMP is a lipid-extracted PMP (LPMP). As used herein, the terms "lipid extraction PMP" and "LPMP" are derived from plant origin (eg, enriched, isolated or purified) lipid structures (eg, lipid bilayers, monolayers, etc.). PMPs derived from multi-layered structures; eg, follicular lipid structures), the lipid structure of which has been disrupted (eg, disrupted by lipid extraction) and using standard methods, eg, as described herein. PMPs reassembled or reconstituted within a lipid layer (eg, a lipid layer containing a cargo) reconstituted using methods comprising lipid membrane hydration and / or solvent injection to produce LPMPs. Point to. The method may further include sonication, freezing / thawing and / or lipid extrusion, if desired, for example to reduce the particle size of the reconstituted PMP. PMPs (eg, LPMPs) can contain 10% to 100% lipids derived from plant-derived lipid structures, eg, at least 10%, at least 20%, at least 30%, at least 40 from plant-derived lipid structures. %, At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of lipids. PMPs (eg, LPMPs) may comprise all or a fraction of the lipid species present in plant-derived lipid structures, eg, at least 10%, at least 20%, at least in plant-derived lipid structures. It may contain 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% lipid species. PMPs (eg, LPMPs) may contain no, fractions or all of the protein species present in the lipid structure of plant origin, eg 0%, less than 1%, less than 5%, less than 10%, 15 Less than%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100% or 100% in lipid structures derived from plant origin May include existing protein species. In some examples, the lipid bilayer of PMP (eg, LPMP) does not contain protein. In some examples, the lipid structure of PMP (eg, LPMP) contains a reduced amount of protein compared to the lipid structure of plant origin.

PMP (eg, LPMP) is optionally described as an exogenous lipid, eg, (1) (eg, originating from a plant or non-plant part from which PMP is produced) (eg, described herein). It is either exogenous to the plant (added to the PMP using the method) or (2) endogenous to the plant cell or tissue from which the PMP is produced, but higher than naturally found (eg, natural). Was it present in PMP at a concentration (higher than that found in plant extracellular vesicles present in) (eg, added to PMP using the methods described herein, genetic engineering, in vitro or in vivo approaches)? Contains lipids that are any of the above. The lipid composition of PMP is 0%, less than 1% or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. , 80%, 90%, 95% or more than 95% foreign lipids. Typical foreign lipids include cationic lipids, ionizable lipids, zwitterionic lipids and lipidoids.

The PMP may optionally include additional agents such as heterologous functional agents such as plant modifiers, agricultural agents, fertilizers, polynucleotides, polypeptides or small molecules. PMP can be used in a variety of ways to allow delivery of a drug to a target plant, such as encapsulation of the drug, incorporation of the drug into a lipid bilayer structure, or binding of the surface of the lipid bilayer structure to the drug (eg, conjugation). ) Can carry or bind additional agents (eg, plant modifiers). Heterologous agents can be integrated into PMP either in vivo (eg, in plants) or in vitro (eg, in tissue cells, in cell cultures or synthetically incorporated).

As used herein, the term "cationic lipid" refers to an amphipathic molecule (eg, lipid or lipidoid) containing a cationic group (eg, a cationic head group).

As used herein, the term "lipidoid" refers to a molecule that has one or more properties of a lipid.

As used herein, the term "plant modifier" alters genetic properties (eg, increases gene expression, reduces gene expression, or otherwise nucleotide sequences of DNA or RNA. A drug that can alter the biochemical properties of a plant in a manner that can (alter) or cause an increase in the adaptability of the plant.

As used herein, the term "stable PMP composition" (eg, a composition comprising loaded or non-loaded PMP) is used for a period of time (eg, at least 24 hours, at least 48 hours, at least 1 week). Optional defined temperature range (eg, at least 24 ° C, 25 ° C, for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days or at least 90 days). 26 ° C, 27 ° C, 28 ° C, 29 ° C or 30 ° C), at least 20 ° C (eg at least 20 ° C, 21 ° C, 22 ° C or 23 ° C), at least 4 ° C (eg at least 5 ° C, 10 ° C or 15). ° C), at least -20 ° C (eg, at least -20 ° C, -15 ° C, -10 ° C or 0 ° C) or at least -80 ° C (eg, at least -80 ° C, -70 ° C, -60 ° C, -50 ° C). , −40 ° C. or −30 ° C.)), the initial number of PMPs (eg, PMPs per mL of solution) compared to the number of PMPs in the PMP composition (eg, at the time of production or formulation). At least 5% (eg, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, Keep 75%, 80%, 85%, 90%, 95% or 100%; or optionally specify a temperature range (eg, at least 24 ° C (eg, at least 24 ° C, 25 ° C, 26 ° C). , 27 ° C, 28 ° C, 29 ° C or 30 ° C), at least 20 ° C (eg, at least 20 ° C, 21 ° C, 22 ° C or 23 ° C), at least 4 ° C (eg, at least 5 ° C, 10 ° C or 15 ° C). , At least −20 ° C. (eg, at least −20 ° C., −15 ° C., −10 ° C. or 0 ° C.) or at least −80 ° C. (eg, at least −80 ° C., −70 ° C., −60 ° C., −50 ° C., − At a temperature of 40 ° C. or −30 ° C.), at least 5% (eg, pesticide and / or repellent activity) of PMP (eg, pesticide and / or repellent activity) compared to the initial activity of PMP (eg, at the time of production or formulation). , At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, Refers to a PMP composition that retains 85%, 90%, 95% or 100%).

As used herein, the term "untreated" refers to plants that have not been contacted or delivered to the plant modification composition, including individual plants to which the plant modification composition has not been delivered. The same plant treated is evaluated before delivery of the plant modification composition, or the same plant treated is evaluated in the untreated portion of the plant.

As used herein, the term "juice sac" or "juice vesicles" refers to the juice-containing membrane-binding element of citrus-like fruits, such as the inner fruit bark (carpel) of citrus fruits. .. In some embodiments, the sandpin is separated from other parts of the fruit, such as the skin (exodermis or flaved), endodermis (mesicarp, albedo or pith), stele (placenta), split wall or seed. In some embodiments, the vesicles are grapefruit, lemon, lime or orange vesicles.

FIG. 6 illustrates a protocol for grapefruit PMP production using a destructive squeezing step involving the use of blender, followed by ultracentrifugation and sucrose gradient purification. Images of grapefruit juice after centrifugation at 1000 xg for 10 minutes and sucrose gradient band pattern after ultracentrifugation at 150,000 xg for 2 hours are included. It is a plot of the PMP particle size distribution measured by Spectradyne NCS1. FIG. 6 illustrates a protocol for grapefruit PMP production using a gentle squeezing step involving the use of a mesh filter, followed by centrifugation and sucrose gradient purification. Images of grapefruit juice after centrifugation at 1000 xg for 10 minutes and sucrose gradient band pattern after ultracentrifugation at 150,000 xg for 2 hours are included. FIG. 6 illustrates a protocol for grapefruit PMP production using ultracentrifugation followed by size exclusion chromatography (SEC) for isolating PMP-containing fractions. The eluted SEC fractions are analyzed for particle concentration (NanoFCM), median particle size (NanoFCM) and protein concentration (BCA). FIG. 6 is a graph showing particle concentration per mL in an eluted size exclusion chromatography (SEC) fraction (NanoFCM). The fraction containing most of the PMP (“PMP fraction”) is indicated by an arrow. PMP is eluted in fractions 2-4. A series of graphs and tables showing particle size (nm) for selected SEC fractions, as measured using NanoFCM. The graph shows the PMP particle size distribution of fractions 1, 3, 5 and 8. It is a graph which shows the protein concentration (μg / mL) in the SEC fraction measured by using the BCA assay. The fraction containing most of the PMP (“PMP fraction”) is labeled and the arrows indicate the fractions containing contaminants. Use a juice squeezer, followed by fractional centrifugation to remove large debris, 100x concentration of juice using TFF and size exclusion chromatography (SEC) to isolate PMP-containing fractions, 1 FIG. 6 is a schematic showing a protocol for scaled PMP generation from liters of grapefruit juice (about 7 grapefruits). The SEC elution fraction is analyzed for particle concentration (NanoFCM), median particle size (NanoFCM) and protein concentration (BCA). Scale of 1000 ml Grapefruit Juice A pair of graphs showing protein concentration (BCA assay, upper panel) and particle concentration (NanoFCM, lower panel) of SEC eluate volume (ml) from starting material, late SEC elution. It shows a large amount of contaminants in volume. Incubation of a crude grapefruit PMP fraction with a final concentration of 50 mM EDTA, pH 7.15, followed by overnight dialysis with a 300 kDa membrane, contamination present in the late SEC-eluted fraction, as indicated by absorbance at 280 nm. It is a graph which shows that the removal of an object was successful. There was no difference in the dialysis buffers used (PBS without calcium / magnesium, pH 7.4, MES pH 6, Tris pH 8.6). By BCA protein analysis (which can detect the presence of sugar and pectin in addition to protein detection) by incubation of a crude grapefruit PMP fraction with a final concentration of 50 mM EDTA, pH 7.15, followed by overnight dialysis with a 300 kDa membrane. As shown, it is a graph showing that the contaminants present in the late elution fraction after SEC were successfully removed. There was no difference in the dialysis buffers used (PBS without calcium / magnesium, pH 7.4, MES pH 6, Tris pH 8.6). As measured by nanoflow cytometry (NanoFCM), it is a graph showing the particle concentration (particles / ml) in the SEC fraction of the eluted BMS plant cell culture. PMP was eluted in SEC fractions 4-6. FIG. 3 is a graph showing the absorbance (AU) at 280 nm in an eluted BMS SEC fraction as measured by a SpectraMax® spectrophotometer. PMP was eluted in fractions 4-6; fractions 9-13 contained contaminants. It is a graph which shows the protein concentration (μg / ml) in the elution BMS SEC fraction as determined by BCA analysis. PMP was eluted in fractions 4-6; fractions 9-13 contained contaminants. It is a scatter plot which shows the particle in the combined BMS PMP containing SEC fraction as measured by nanoflow cytometry (NanoFCM). The PMP concentration (particles / ml) was determined using a bead standard according to the instructions of NanoFCM. It is a graph which shows the particle size distribution (nm) of the BMS PMP of the gated particle (background removal) of FIG. 5D. The median PMP particle size (nm) was determined using the Exo bead standard according to the instructions of NanoFCM. Scatter plots and graphs showing particle size of AF488-labeled lemon PMP as measured by nanoflow cytometry (NanoFCM). The upper panel is a scatter plot showing AF488-labeled lemon PMP. The particles were gated for FITC fluorescent signals against unlabeled particles and background signals. The labeling efficiency was 89.4%, as determined by the number of fluorescent particles relative to the total number of detected particles. ^{The final AF488-PMP concentration (2.91 × 10 12} PMP / ml) was determined from the number of fluorescent particles and using a bead standard of known concentration according to the instructions of NanoFCM. The lower panel is a particle size (nm) distribution graph of AF488-labeled lemon PMP. The median PMP particle size was determined using the Exo bead standard according to the instructions of NanoFCM. The median lemon PMP particle size was 79.4 nm +/- 14.7 nm (SD). Plant cell line: Alexa Fluor® 488 (AF488) labeled lemon (LM) with Glycine max (soybean), Triticum aestivum (wheat) and corn BMB cell culture. It is a series of micrographs showing the uptake of PMP. The brightfield panel shows the location of the cells; the panel labeled with "GFP" shows the fluorescence of AF488. Uptake of PMP by cells is indicated by the presence of AF488 in cells. Free AF488 (“free dye”) is shown as a control. A pair of schematics and a series of micrographs showing the uptake of DL800-labeled lemon (LM) and grapefruit (GF) PMP by Arabidopsis thaliana seedlings and alfalfa sprout. The fluorescence intensity of the DL800 dye is displayed. Fluorescence intensity was measured at 22 hpt (at the time point after treatment) for Arabidopsis thaliana seedlings and 24 hpt for alfalfa sprout. Seedlings incubated without dye (“negative control”) and with free DL800 dye (“DL800 single control”) are shown as controls. It is a figure which shows the experimental outline about the treatment of alfalfa sprout using the DyLight 800 nm labeled PMP produced with or without pectinase treatment. An infrared heating map showing the uptake of purified lemon PMP in alfalfa sprout, showing that pectinase treatment during lemon PMP production, followed by DL800 nm labeling of the purified PMP, does not affect PMP uptake or transport. It is a figure which shows. The infrared image is taken on the Odyssey scanner. Two-week-old Arabidopsis thaliana seedlings stained with DiR (LM-LPMP-DiR), untreated (untreated) or treated with lemon (LM) lipid rearrangement PMP (LPMP) It is a figure which shows the pair of the micrograph which shows the fluorescence of the DiR dye at 750 nm. Images were acquired with an iBright 1500 fluorescence imager. Micrograph showing fluorescence of DiR dye at 750 nm in 2 week old Arabidopsis thaliana seedlings treated with Grapefruit (GF) LPMP stained with DiR control or DiR (GF-LPMP-DiR) It is a figure which shows the pair of photographs. Images were acquired with an iBright 1500 fluorescence imager. Using LPMP loaded with equimolar amounts of FWA-gRNA 4 and 17 (LPMP sgRNA) and LPMP loaded with DC-cholesterol loaded with equimolar amounts of FWA-gRNA 4 and 17 (DC-Chol sgRNA). FIG. 6 is a bar graph showing RT-qPCR gene expression analysis of FLOWERING WAGENINGEN (FWA) compared to ubiquitin (UBI) in Arabidopsis thaliana dCas9-SunTag-VP64 plants treated for 48 hours. LPMP supplemented with an equal amount of duplicated FWA-gRNA 4/17 mixture (sgRNA4 / 17) and DC-cholesterol loaded with only tracrRNA (without gRNA) is provided as a control. The data are presented as mean ± SD. ^* One-way ANOVA with p <0.05, Sidak post-hook test. LPMP loaded with equimolar amounts of FWA-gRNA 4 and 17 (LPMP sgRNA) and equimolar amounts of FWA-gRNA 4 and 17 (DC-Chol sgRNA) measured using RT-qPCR gene expression analysis. FWA compared to a double FWA-gRNA 4/17 mixture (sgRNA4 / 17) control in Arabidopsis thaliana dCas9-SunTag-VP64 plants treated with LPMP supplemented with loaded DC-cholesterol for 48 hours. It is a bar graph which shows the induction magnification of. The data are presented as mean ± SD. ^* One-way ANOVA with p <0.05, Sidak post-hook test. LPMP loaded with equimolar amounts of FWA-gRNA 4 and 17 (LPMP sgRNA) and equimolar amounts of FWA-gRNA 4 and 17 (DC-Chol sgRNA) measured using RT-qPCR gene expression analysis. ATISOPENTEYL dirin compared to a double FWA-gRNA 4/17 mixture (sgRNA4 / 17) control in Arabidopsis thaliana dCas9-SunTag-VP64 plants treated with LPMP with loaded DC-cholesterol for 48 hours. It is a bar graph which shows the induction magnification of acid isomerase 2 (IPP2). The data are presented as mean ± SD. ^* One-way ANOVA with p <0.05, Sidak post-hook test. It is a schematic diagram which shows the workflow for preparing a lipid rearrangement PMP (LPMP) from a grapefruit and a lemon PMP. LPMP; LPMP to which DC-cholesterol (DC-Chole) is added; and LPMP to which DOTAP (DOTAP) is added are graphs showing the relative frequency of particles having a predetermined particle size (nm). Data were obtained by NanoFCM using the concentration and particle size criteria provided by the manufacturer. It shows the zeta potential (mV) of LPMP without added lipids (LPMP) and LPMP with 25% or 40% DOTAP or DC-cholesterol measured using dynamic light scattering (DLS). It is a graph. The data are presented as mean ± SD. Bar graph showing% of Alexa Fluor 555 labeled siRNA input recovered from LPMP after loading LPMP from grapefruit lipid without added lipid (LPMP) and LPMP from grapefruit lipid with 20% DOTAP. Is. Bar graph showing% of ATTO-labeled TracrRNA input recovered from LPMP after loading of LPMP derived from lemon lipid without added lipid (LPMP) and LPMP derived from lemon lipid containing 40% DC-cholesterol. Is. TracrRNA in LPMP containing 40% DC-cholesterol untreated or dissolved with Triton-X100 and heparin (+ TX + heparin) as measured using Quant-iT ™ RiboGreen® analysis. It is a bar graph which shows the concentration (μg / mL). It is a cryo-electron micrograph showing LPMP reconstituted from the extracted lemon lipid. Scale bar: 500 nm. 6 is a graph showing the relative frequency of particles of predetermined equivalent sphere diameter (nm) in LPMP reconstituted from extracted lemon lipids, measured using cryo-electron micrographs. Phase difference (left column) of corn Black Mexican Sweet (BMS) cells treated with LPMP (middle row) without lipids and LPMP (DC-Chol) with 40% DC-cholesterol, ATTO. It is a series of micrographs showing 550 fluorescence (middle column) and combined images. H 2 _O alone treated cells was provided as a negative control (upper panel). Cellular uptake of LPMP or DC-cholesterol-modified LPMP is dictated by the presence of the intracellular TracrRNA ATTO 550 signal. Scale bar: 100 μm.

Characterized herein are plant messenger packs (PMPs), plant modifications containing whole or part of plant extracellular vesicles (EVs) or lipid aggregates produced from fragments, parts or extracts thereof. Compositions and related methods for modifying plants (eg, increasing plant adaptability) using the compositions. The PMPs provided herein include plant modifiers. Also included are plant-modified formulations in which PMP is delivered in substantially pure or concentrated form. The plant modification compositions and formulations described herein are used to modify plants in a manner that introduces (eg, increases the adaptability of the plant) the desired plant traits in plants such as agricultural or garden plants. It can be delivered directly to the plant.

I. Plant Modification Compositions The plant modification compositions described herein include multiple plant messenger packs (PMPs). PMP is a lipid (eg, lipid bilayer, monolayer or multi-layer structure) structure containing plant EV or fragments, portions or extracts thereof (eg, lipid extracts). Plant EV refers to an encapsulated lipid bilayer structure naturally occurring in plants, having a diameter of about 5 to 2000 nm. Plant EVs can arise from a variety of plant biogenic pathways. In fact, plant EVs can be found in the intracellular and extracellular compartments of plants such as plant apoplasts, which are located outside the original plasma membrane and are compartments formed by the cell wall and extracellular space continuum. Alternatively, the PMP can be a concentrated plant EV found in cell culture medium when secreted from plant cells. Plant EVs can be secreted from plants (eg, from apoplast fluid), which can further generate PMP by various methods described herein. The PMP herein further comprises a heterologous plant modifier that can be introduced using various in vivo or in vitro methods such as those described further herein.

PMPs can include plant EVs or fragments, portions or extracts thereof. Optionally, the PMP can further include foreign lipids in addition to the lipids derived from plant EV. In some embodiments, the plant EV is about 5 to 2000 nm in diameter. For example, PMP is about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, About 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, It may contain a plant EV or a fragment, portion or extract thereof having an average diameter of about 950 to 1000 nm, about 1000 to 1250 nm, about 1250 to 1500 nm, about 1500 to 1750 nm or about 1750 to 2000 nm. In some examples, the PMP is about 5 to 950 nm, about 5 to 900 nm, about 5 to 850 nm, about 5 to 800 nm, about 5 to 750 nm, about 5 to 700 nm, about 5 to 650 nm, about 5 to 600 nm, about. 5 to 550 nm, about 5 to 500 nm, about 5 to 450 nm, about 5 to 400 nm, about 5 to 350 nm, about 5 to 300 nm, about 5 to 250 nm, about 5 to 200 nm, about 5 to 150 nm, about 5 to 100 nm, about. Contains a plant EV or a fragment, portion or extract thereof having an average diameter of 5-50 nm or about 5-25 nm. In certain examples, the plant EV or a fragment, portion or extract thereof has an average diameter of about 50-200 nm. In certain examples, the plant EV or a fragment, portion or extract thereof has an average diameter of about 50-300 nm. In certain examples, the plant EV or a fragment, portion or extract thereof has an average diameter of about 200-500 nm. In certain examples, the plant EV or fragments, portions or extracts thereof have an average diameter of about 30-150 nm.

In some examples, the PMP is at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm. Can contain plant EVs or fragments, portions or extracts thereof having an average diameter of at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm or at least 1000 nm. In some examples, the PMP is less than 1000 nm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm. Contains plant EVs or fragments, portions or extracts thereof having an average diameter of less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm or less than 50 nm. Various methods standard in the art (eg, dynamic light scattering) can be used to measure the particle size of plant EVs or fragments, portions or extracts thereof.

In some instances, PMP ^{is, 77nm 2 ~3.2 × 10 6 nm} 2 ( ^{e.g., 77~100nm 2, 100~1000nm 2, 1000~1} × 10 4 nm 2, 1 × 10 4 ~1 × 10 ^May contain plant EV or fragments, portions or extracts thereof having an average surface area of 5 nm ² , 1 × 10 ⁵ to 1 × 10 ⁶ nm ² or 1 × 10 ^{6 to} 3.2 × 10 ⁶ nm ^2). .. In some instances, PMP ^{is, 65nm 3 ~5.3 × 10 8 nm} 3 ( ^{e.g., 65~100nm 3, 100~1000nm 3, 1000~1} × 10 4 nm 3, 1 × 10 4 ~1 × 10 ⁵ nm ³ , 1 × 10 ⁵ to 1 × 10 ⁶ nm ² , 1 × 10 ⁶ to 1 × 10 ⁷ nm ³ , 1 × 10 ⁷ to 1 × 10 ⁸ nm ³ , 1 × 10 ^{8 to} 5.3 × 10 ^It may contain a plant EV or a fragment, portion or extract thereof having an average volume of 8 nm ^3). In some examples, the PMP is at least 77 nm ² (eg, at least 77 nm ² , at least 100 nm ² , at least 1000 nm ² , at least 1 × 10 ⁴ nm ² , at least 1 × 10 ⁵ nm ² , at least 1 × 10 ⁶ nm ^2). Alternatively, it may contain a plant EV or a fragment, portion or extract thereof having an average surface area of at least 2 × 10 ⁶ nm ^2). In some examples, the PMP is at least 65 nm ³ (eg, at least 65 nm ³ , at least 100 nm ³ , at least 1000 nm ³ , at least 1 × 10 ⁴ nm ³ , at least 1 × 10 ⁵ nm ³ , at least 1 × 10 ⁶ nm ^3). , At least 1 × 10 ⁷ nm ³ , at least 1 × 10 ⁸ nm ³ , at least 2 × 10 ⁸ nm ³ , at least 3 × 10 ⁸ nm ³ , at least 4 × 10 ⁸ nm ³ or at least 5 × 10 ⁸ nm ³ ) It may contain a plant EV or a fragment, portion or extract thereof having an average volume.

In some examples, the PMP can have the same size as a plant EV or a fragment, extract or portion thereof. Instead, the PMP may have a different size than the early plant EV from which the PMP is produced. For example, PMP can have a diameter of about 5 to 2000 nm. For example, PMP is about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, About 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, It can have an average diameter of about 950 to 1000 nm, about 1000 to 1200 nm, about 1200 to 1400 nm, about 1400 to 1600 nm, about 1600 to 1800 nm or about 1800 to 2000 nm. In some examples, the PMP is at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm. Can have an average diameter of at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm or at least 2000 nm. Various methods standard in the art (eg, dynamic light scattering) can be used to measure the particle size of PMP. In some examples, the size of the PMP is determined after loading the heterologous plant modifier or after other modifications to the PMP.

In some instances, PMP ^{is, 77nm 2 ~1.3 × 10 7 nm} 2 ( ^{e.g., 77~100nm 2, 100~1000nm 2, 1000~1} × 10 4 nm 2, 1 × 10 4 ~1 × 10 ^It can have an average surface area of 5 nm ² , 1 × 10 ⁵ to 1 × 10 ⁶ nm ² or 1 × 10 ^{6 to} 1.3 × 10 ⁷ nm ^2). In some instances, PMP ^{is, 65nm 3 ~4.2 × 10 9 nm} 3 ( ^{e.g., 65~100nm 3, 100~1000nm 3, 1000~1} × 10 4 nm 3, 1 × 10 4 ~1 × 10 ⁵ nm ³ , 1 × 10 ⁵ to 1 × 10 ⁶ nm ² , 1 × 10 ⁶ to 1 × 10 ⁷ nm ³ , 1 × 10 ⁷ to 1 × 10 ⁸ nm ³ , 1 × 10 ⁸ to 1 × 10 ⁹ nm ^It can have an average volume of 3 or 1 × 10 ^{9 to} 4.2 × 10 ⁹ nm ^3). In some examples, the PMP is at least 77 nm ² (eg, at least 77 nm ² , at least 100 nm ² , at least 1000 nm ² , at least 1 × 10 ⁴ nm ² , at least 1 × 10 ⁵ nm ² , at least 1 × 10 ⁶ nm ^2). Or it has an average surface area of 1 × 10 ⁷ nm ^3). In some examples, the PMP is at least 65 nm ³ (eg, at least 65 nm ³ , at least 100 nm ³ , at least 1000 nm ³ , at least 1 × 10 ⁴ nm ³ , at least 1 × 10 ⁵ nm ³ , at least 1 × 10 ⁶ nm ^3). , At least 1 × 10 ⁷ nm ³ , at least 1 × 10 ⁸ nm ³ , at least 1 × 10 ⁹ nm ³ , at least 2 × 10 ⁹ nm ³ , at least 3 × 10 ⁹ nm ³ or at least 4 × 10 ⁹ nm ³ ) Has an average volume.

In some examples, the PMP may contain intact plant EVs. Instead, PMP is a fragment, portion or extract of the total surface area of the vesicles of the plant EV (eg, less than 100% of the total surface area of the vesicles (eg, less than 90%, less than 80%, less than 70%, 60%). Can contain fragments, moieties or extracts) containing less than, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1%). The fragment, portion or extract can be of any shape, such as a circumferential fragment, a spherical fragment (eg, a hemisphere), a curved fragment, a linear fragment or a flat plate fragment. If the fragment is a spherical fragment of vesicles, the spherical fragment results from the division of spherical vesicles along a pair of parallel lines or from the division of spherical vesicles along a pair of non-parallel lines. Can be presented. Thus, multiple PMPs can include multiple intact plant EVs, multiple plant EV fragments, partials or extracts or mixtures of intact plant EVs and fragments thereof. Those skilled in the art will appreciate that the ratio of intact and fragmented plant EVs will vary depending on the specific isolation method used. For example, grinding or blending of plants or portions thereof can produce PMPs containing higher proportions of plant EV fragments, moieties or extracts than non-destructive extraction methods such as vacuum infiltration.

In an example where the PMP contains a fragment, moiety or extract of plant EV, the EV fragment, moiety or extract has a smaller average surface area of the intact vesicles, eg 77 nm ² , 100 nm ² , 1000 nm ² , 1 ×. It can have an average surface area of less than ^{10 4} nm ² , 1 × 10 ⁵ nm ² , 1 × 10 ⁶ nm ² or 3.2 × 10 ⁶ nm ^2). In some examples, the EV fragment, moiety or extract has a surface area of less than ^{70 nm 2} , 60 nm ² , 50 nm ² , 40 nm ² , 30 nm ² , 20 nm ² or 10 nm ^2). In some examples, the PMP has a smaller average volume of intact vesicles, such as 65 nm ³ , 100 nm ³ , 1000 nm ³ , 1 × 10 ⁴ nm ³ , 1 × 10 ⁵ nm ³ , 1 × 10 ⁶ nm ^3. It may contain a plant EV or a fragment, portion or extract thereof having an average volume of less than 1, 1 × 10 ⁷ nm ³ , 1 × 10 ⁸ nm ³ or 5.3 × 10 ⁸ nm ^3).

In examples where the PMP comprises an extract of plant EV, eg, PMP comprises a lipid extracted from plant EV (eg, in chloroform), the PMP comprises a lipid extracted from plant EV (eg, in chloroform). Contains at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more than 99%. obtain. A large number of PMPs may contain plant EV fragments and / or plant EV extracted lipids or mixtures thereof.

This specification further outlines the method for producing PMP, the preparation of the composition containing the plant EV marker and PMP that can be bound to PMP.

A. Production Method PMP can be produced from a plant EV or a fragment, portion or extract thereof (eg, a lipid extract) that is naturally present in the plant or its portion containing plant tissue or plant cells. An exemplary method of producing PMP is (a) a step of providing an initial sample from a plant or portion thereof, wherein the plant or portion thereof comprises an EV; (b) a crude PMP picture from the initial sample. A step of isolating a minute, wherein the crude PMP fraction has at least one contaminant or unwanted component from the plant or portion thereof at a reduced level relative to the level in the initial sample. including. This method is an additional step (c) of purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs are reduced relative to the level of the crude PMP fraction. It may further comprise an additional step (c) with at least one contaminant or unwanted component from the plant or portion thereof at the indicated level. Each generation step is discussed in more detail below. Exemplary methods for isolation and purification of PMP are described, for example, in Rutter and Innes, Plant Physiol. 173 (1): 728-741, 2017; Rutter et Al, Bio. Protocol. 7 (17): e2533, 2017; Regent et Al, Job Exp. Biol. 68 (20): 5485-5494, 2017; Mu et Al, Mol. Nutr. Food Res. , 58, 1561-1573, 2014 and Regent et Al, FEBS Letters. 583: 3363-3366, 2009, each of which is incorporated herein by reference.

In some examples, (a) a step of providing an initial sample from a plant or portion thereof, wherein the plant or portion thereof comprises an EV; (b) isolating a crude PMP fraction from the initial sample. The crude PMP fraction is a reduced level (eg, at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30) relative to the level in the initial sample. %, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100% reduced levels) of the plant or portion thereof. A step comprising at least one contaminant or unwanted component from; (c) a step of purifying a crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs are: Reduced levels for crude EV fractions (eg at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55% , 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100% reduced levels) with at least one contaminant or unwanted component from the plant or parts thereof. Multiple PMPs can be isolated from the plant by a process involving steps.

The PMPs provided herein may include plant EVs or fragments, portions or extracts thereof produced from a variety of plants. PMPs can be produced from plants of any genus (tubal or non-tubal bundles) and include, but are not limited to, subplants (single and dicotyledonous plants), pterophyta, pterophyta, selaginella. ), Selaginella, pterophytes, lycophyte, algae (eg, single-cell or multi-cell, such as archaeplastida) or moss. In certain examples, PMP can be produced using vascular plants such as monocotyledons or dicotyledons or gymnosperms. For example, PMPs include alfalfa, apples, rapeseed, bananas, corns, canolas, sesame seeds, chicory, kiku, clover, cacao, coffee, cotton, cottonseed, corn, cranbe, cranberries, cucumbers, dendrobium, yamaimo, eucalyptus. Genus, bovine rape, flax, gradiolas, lily family, flaxseed, millet, mask melon, rapeseed, oatsumugi, oilseed rape, oilseed rape, papaya, peanuts, pineapple, ornamental plants, green beans, potatoes, rapeseed, rice, limewood, ryegrass, rapeseed, sesame , Morokoshi, soybean, tensai, sugar cane, sunflower, strawberry, tobacco, tomato, turfgrass, wheat or lettuce, celery, broccoli, potash flower, vegetable crops such as rapeseed; apple, pear, peach, orange, grapefruit, lemon, lime , Almonds, pecans, walnuts, hazel and other fruits and fruit trees; grapes, kiwi, hops and other fruits; raspberries, blackberries, oilseed rape and other fruit shrubs and strawberry; , Poplar and other forest trees; alfalfa, canola, corn, corn, cotton, crumb, flax, flaxseed, mustard, rapeseed, rape, peanut, potato, rice, benibana, sesame, soybean, tensai, sunflower, tobacco or wheat Can be generated.

PMP uses whole plants (eg whole rosettes or seedlings) or alternative plant parts (eg leaves, seeds, roots, fruits, vegetables, pollen, phloem sap or xylem sap). Can be generated. For example, PMPs are sprout plant organs / structures (eg, leaves, stems or ovules), roots, flowers and flower vessels / structures (eg, pollen, follicles, stalks, petals, phloems, carpels, anthers or ovules), seeds. (Including embryos, endosperm or seed coat), fruits (mature ovules), sap (eg, phloem or wood sap), plant tissues (eg, ductal bundle tissue, basic tissue, tumor tissue, etc.) and cells (eg, tubule tissue) , Single cell, protoplast, embryo, callus tissue, phloem cell, egg cell, etc.) or their progeny. For example, the isolation step may include (a) preparing a plant or a portion thereof, where the plant portion is a leaf of Arabidopsis. The plant can be of any stage of development. For example, PMP is produced using seedlings such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks old seedlings (eg Arabidopsis seedlings). be able to. Other exemplary PMPs are roots (eg, root ginger), fruit juice (eg, grapefruit juice), vegetables (eg, broccoli), pollen (eg, olive pollen), phloem fluid (eg, Arabidopsis) masters. It may contain PMP produced using phloem) or xylem sap (eg, tomato plant xylem sap).

PMP can be produced using a plant or parts thereof by various methods. Any method that allows the release of an EV-containing apoplast fraction of a plant or an extracellular fraction containing a PMP containing otherwise secreted EV (eg, cell culture medium) is suitable in this method. EV is separated from the plant or plant part by either destructive (eg, grinding or blending of the plant or any plant part) or non-destructive (cleaning or vacuum infiltration of the plant or any plant part) method. Can be done. For example, a plant or portion thereof can be subjected to vacuum infiltration, grinding, blending or a combination thereof to isolate the EV from the plant or plant portion, thereby producing PMP. For example, the step of isolation is (b) the step of isolating the crude PMP fraction from an initial sample (eg, a plant, plant portion or sample derived from a plant or plant portion), wherein the crude PMP fraction is: It may comprise a step with at least one contaminant or unwanted component from the plant or portion thereof, at a reduced level relative to the level in the initial sample; the step of isolation may include plant (eg, vesicle isolation). Includes vacuum infiltration (with a buffer) to release and collect the apoplast fraction. Alternatively, the isolation step can include grinding or blending the plant to release EV, thereby producing PMP.

Once the plant EV is isolated and thereby produced PMP, the PMP can be collected in a separated or crude PMP fraction (eg, apoplast fraction). For example, the separation step is centrifugation (eg, fractional centrifugation) to separate plant PMP-containing fractions from large contaminants containing plant necrotic tissue, plant cells or plant cell organellas (eg, nuclei or chloroplasts). Separation or ultracentrifugation) and / or may include the step of separating multiple PMPs into crude PMP fractions using filtration. Therefore, the crude PMP fraction will reduce the number of large contaminants, including plant necrotic tissue or plant cells, compared to early samples from the plant of origin or plant part. Depending on the method used, the crude PMP fraction adds reduced levels of plant cell organelles (eg, nuclei, mitochondria or chloroplasts) compared to early samples from the plant of origin or plant part. Can include.

In some examples, the isolation step uses centrifugation (eg, fractional centrifugation or ultracentrifugation) and / or filtration to separate plant EV-containing fractions from plant cells or plant tissue debris. , May include separating multiple PMPs into coarse PMP fractions. Therefore, the crude PMP fraction will reduce the number of plant cell or plant tissue debris compared to the initial sample from the plant of origin or plant portion.

The crude PMP fraction can also be further purified by additional purification methods to produce multiple pure PMPs. For example, by ultracentrifugation, for example, by using a density gradient (iodixanol or sucrose) and / or by using other techniques for removing aggregate elements (eg, precipitation or size exclusion chromatography), the crude PMP fraction. Can be separated from other plant elements. The resulting pure PMP is contaminated with one or more fractions or predetermined threshold levels produced during the earlier separation steps, eg, reduced levels for commercial publication specifications, from the plant of origin. A substance or unwanted component (eg, one or more non-PMP components such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures), nuclei, cell wall components, cell organellas or These combinations) may be possessed. For example, pure PMP is reduced levels relative to the levels of the initial sample (eg, about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80). %, 90%, 100% or more than 100%; or about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x or more than 100x) plant organelles or cell walls. May have ingredients. In some examples, pure PMP is one such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures), nuclei, cell wall components, cell organellas or combinations thereof. Alternatively, it is substantially free of multiple non-PMP components (eg, having undetectable levels thereof). Yet another example of the release and separation steps can be found in Example 1. PMP is, for example, 1 × 10 ⁹ , 5 × 10 ⁹ , 1 × 10 ¹⁰ , 5 × 10 ¹⁰ , 5 × 10 ¹⁰ , 1 × 10 ¹¹ , 2 × 10 ¹¹ , 3 × 10 ¹¹ , 4 × 10 ¹¹ . 5x10 ¹¹ , 6x10 ¹¹ , 7x10 ¹¹ , 8x10 ¹¹ , 9x10 ¹¹ , 1x10 ¹² , 2x10 ¹² , 3x10 ¹² , 4x10 ¹² , 5x10 ¹² , ^{6 × 10 12, 7 × 10} 12, 8 × 10 12, 9 × 10 12, 1 × 10 13 or 1 × ¹⁰ 13 can be a concentration of PMPs / mL greater.

For example, protein aggregates can be removed from PMP. For example, PMP can be applied to various pH (eg, as measured with a pH probe) to precipitate protein aggregates in solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9 or pH 11 by adding sodium hydroxide or hydrochloric acid, for example. Once the solution reaches the specified pH, it can be filtered to remove fine particles. Alternatively, PMP can be aggregated by the addition of a charged polymer such as Polymin-P or Prestol 2640. Briefly, Polymin-P or Praestol 2640 is added to the solution and mixed with an impeller. The solution is then filtered to remove the particulates. Alternatively, the aggregates can be solubilized by increasing the salt concentration. For example, NaCl can be added to PMP until it is, for example, 1 mol / L. The solution is then filtered to isolate the PMP. Alternatively, the aggregate can be solubilized by heating. For example, while mixing PMP, the solution can be heated for 5 minutes until it reaches a uniform temperature of, for example, about 50 ° C. The PMP mixture can then be filtered to isolate the PMP. Alternatively, size exclusion chromatography according to standard procedures can separate soluble contaminants from the PMP solution, in which case the PMP is eluted during the first fraction, while the protein and ribonucleoprotein as well as some The lipoprotein is later eluted. The efficiency of protein aggregate removal can be determined by measuring and comparing protein concentrations before and after removal of protein aggregates using BCA / Bladeford protein quantification.

Any quantitative or qualitative method known in the art may be added to any of the production methods described herein to characterize or identify PMP at any step of the production method. PMP can be characterized by a variety of analytical methods for estimating PMP yield, PMP concentration, PMP purity, PMP composition or PMP size. PMPs are several methods known in the art that allow visualization, quantification or qualitative characterization of PMPs (eg, composition identification), such as microscopy (eg, transmission electron microscopy), dynamics. It can be evaluated by dynamic light scattering, nanoparticle tracing, spectroscopy (eg, Fourier transform infrared spectroscopy) or mass spectrometry (protein and lipid analysis). In certain examples, methods (eg, mass spectrometry) can be used to identify plant EV markers present in PMP, such as those disclosed in the appendix. The PMP can be further labeled or stained to aid in the analysis and characterization of the PMP fraction. For example, 3,3'-dihexyloxacarbocyanine _{iodine (DIOC 6} ), fluorescent lipophilic dye, PKH67 (Sigma Aldrich); Alexa Fluo® 488 (Thermo Fisher Scientific) or DyLight ™ 800 (Ther). ), The PMP can be stained. Even in the absence of highly morphological nanoparticle tracking, this relatively simple technique can be used to indirectly measure the concentration of PMP by quantifying the total membrane content (Rutter and Innes). , Plant Physiol. 173 (1): 728-741, 2017; Rutter et al, Bio. Protocol. 7 (17): e2533, 2017). Nanoparticle tracking can be used for more accurate measurements and for assessing the particle size distribution of PMP.

During the production process, optionally, PMP is at high concentrations relative to EV levels in the control or initial sample (eg, about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60). %, 70%, 80%, 90%, 100% or more than 100%; or about 2 times, 4 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times or more than 100 times. ), PMP can be prepared. PMP is about 0.1% to about 100% of the plant modification composition, for example about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about. It can occupy any one of 1% to about 25%, about 10% to about 50%, or about 50% to about 99%. In some examples, the composition is measured, for example, by wt / vol, percent PMP protein composition and / or percent lipid composition (eg, by measurement of fluorescently labeled lipids); see, eg, Example 3. ), At least 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% Includes either or more PMPs. In some examples, concentrated agents such as concentrated commercial products are used, for example, the end user may dilute the commercial product, which has a substantially lower concentration of active ingredient. In some embodiments, the composition is formulated as a plant-modified concentrated formulation, eg, a high concentration, low volume formulation.

Producing PMP using a variety of plants or parts thereof (eg, leaf apoplasts, seed apoplasts, roots, fruits, vegetables, pollen, phloem sap or xylem sap) as described in Example 1. Can be done. For example, PMPs can be isolated from plant apoplast fractions, such as leaf apoplasts (eg, Arabidopsis thaliana leaf apoplasts) or seed apoplasts (eg, sunflower seed apoplasts). Other exemplary PMPs include roots (eg, root ginger), fruit juice (eg, grapefruit juice), vegetables (eg, broccoli), pollen (eg, olive pollen), phloem fluid (eg, Arabidopsis). (Tube), xylem sap (eg, tomato plant xylem sap) or cell culture supernatant (eg, BY2 tobacco cell culture supernatant). This example further illustrates the production of PMP from these various plant sources.

As described in Example 2, a density gradient (iodixanol or sucrose) is used by various methods, eg, ultracentrifugation and / or methods for removing aggregate contaminants, eg precipitation or size exclusion chromatography. By doing so, PMP can be purified. For example, Example 2 describes the purification of PMP obtained by the separation step outlined in Example 1. In addition, PMP can be characterized according to the method described in Example 3.

In some examples, the PMP of the composition and method can be produced from a plant or plant portion and can be used without further modification to the PMP. In another example, the PMP can be modified prior to use, as further outlined herein.

B. Plant EV Markers The PMPs of the compositions and methods may have a variety of markers that identify PMPs as produced using plant EVs (and / or fragments, portions or extracts thereof). As used herein, the term "plant EV marker" naturally accompanies a plant and includes plant proteins, plant nucleic acids, plant small molecules, plant lipids or combinations thereof, in or on plant EV in planters. Refers to the elements that are incorporated into. Examples of plant EV markers include, for example, Rutter and Innes, Plant Physiol. 173 (1): 728-741, 2017; Raimondo et al. , Oncotarget. 6 (23): 1951, 2015; Ju et al. , Mol. Therapy. 21 (7): 1345-1357, 2013; Wang et Al. , Molecular Therapy. 22 (3): 522-534, 2014; and Regent et Al, Job Exp. Biol. 68 (20): 5485-5494, 2017; each of these is incorporated herein by reference. Still other examples of plant EV markers are listed in the Appendix, which are further outlined herein.

In some examples, plant EV markers may contain plant lipids. Examples of plant lipid markers that can be found in PMP are phytosterols, campesterols, β-sitosterols, sigmasterols, avenasterols, glycosylinositol phosphorylceramides (GIPCs), glycolipids (eg, monogalactosyldiacylglycerols (MGDGs) or diglycersides). Galactosyldiacylglycerols (DGDGs) or combinations thereof can be mentioned, for example, PMPs can include GIPC, which is representative of the major sphingolipid classes of plants and is one of the most abundant membrane lipids in plants. Other plant EV markers are lipids that accumulate in plants in response to abiotic or biological stressers (eg, bacterial or fungal infections), such as phosphatidic acid (PA) or phosphatidylinositol-4-phosphate (PI4P). ) Can be mentioned.

Alternatively, the plant EV marker may include a plant protein. In some examples, protein plant EV markers can be anti-microbial proteins naturally produced by plants, such as plants in response to abiotic or biological stressors (eg, bacterial or fungal infections). There is a protective protein secreted by. Plant pathogen defense proteins include soluble N-ethylmaleimide susceptibility factor binding protein receptor protein (SNARE) proteins (eg, Syntaxin-121 (SYS121; GenBank accession number: NP_187788.1 or NP_974288.1), Penetration1 (PEN1;). There is a GenBank accession number: NP_567462.1.)) Or an ABC transporter Protein3 (PEN3; GenBank accession number: NP_191283.2). Another example of a plant EV marker is a protein that promotes long-distance transport of RNA in a plant, such as a master protein (eg, master protein 2-A1 (PP2-A1), GenBank accession). Number: NP_193719.1), calcium-dependent lipid-binding protein or lectin (eg, Jacalin-related lectin, eg, Helianthus annuus) Jakarin (Helja; GenBank: AHZ86978.1), for example, RNA-binding protein. Can be a glycine-rich RNA-binding protein-7 (GRP7; GenBank accession number: NP_179760.1). In addition, proteins that regulate plasma phosphatase function are found in plant EV in some examples. However, such are Synap-Totgamin A A (GenBank accession number: NP_565495.1). In some examples, the plant EV marker is involved in the lipid mechanism, such as phosphorlipase C or phosphorlipase D. May include a protein. In some examples, the plant protein EV marker is a plant cell trafficking protein. In certain cases where the plant EV marker is a protein, the protein marker is a signal that typically binds to a secreted protein. Peptides may be deleted. Non-classical secreted proteins are (i) deletion of leader sequence, (ii) absence of ER or Gorgian-specific post-translational modification (PTM), and /. Or (iii) appearing to have some common features, such as unaffected secretion by Brefeldin A, which blocks the classical ER / Gordi-dependent secretory pathway. Those skilled in the art are generally free to use. Proteins can be evaluated for signal sequences or deletions thereof using a variety of possible means (eg, SecretomeP Plantase; SUBA3 (SUBcellular localizati database for Arabidopsis proteinins)).

In the example where the plant EV marker is a protein, the protein is at least 35%, 40%, 45%, 50%, 55%, 60%, 65 with any of the plant EV markers, eg, the plant EV markers listed in the Appendix. It may have an amino acid sequence having a%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity. For example, the proteins are PEN1 (GenBank accession number: NP_567462.1.) Derived from Arabidopsis thaliana and at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, It may have an amino acid sequence with 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity.

In some examples, the plant EV marker comprises a nucleic acid encoded by the plant, such as plant RNA, plant DNA or plant PNA. For example, PMP can include dsRNA, mRNA, viral RNA, microRNA (miRNA) or small interfering RNA (siRNA) encoded by the plant. In some examples, nucleic acids can be related to proteins that promote long-distance transport of RNA in plants, as discussed herein. In some examples, nucleic acid plant EV markers may be involved in host-induced gene silencing (HIGS), which allows plants to carry foreign transcripts of plant pests (eg, pathogens such as fungi). It is a process of suppression. For example, nucleic acids can be those that suppress bacterial or fungal genes. In some examples, the nucleic acid can be a microRNA such as miR159 or miR166 that targets a gene in a fungal pathogen (eg, Verticillium dahliae). In some examples, the protein can be one that is involved in the transport of plant defense compounds, eg, a protein that is involved in glucosinolate (GSL) transport and metabolism, such as the glucosinolate transporter-1-1. (GTR1; GenBank accession number: NP_566896.2), glucosinolate transporter-2 (GTR2; NP_20107.4.1) or epithiospecific modifier 1 (ESM1; NP_188037.1).

In the example where the plant EV marker is a nucleic acid, the nucleic acid is at least 35%, 40%, 45%, 50%, 55%, 60 with a plant EV marker, such as one encoding a plant EV marker listed in the Appendix. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% can have a nucleotide sequence with sequence identity. For example, nucleic acids are at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, with miR159 or miR166. It may have a polynucleotide sequence with 98%, 99% or 100% sequence identity.

In some examples, the plant EV marker comprises a compound produced by the plant. For example, the compound can be a protective compound produced in response to an abiotic or biological stressor, such as a secondary metabolite. One such secondary metabolite that can be found in PMP is glucosinolate (GSL), which is a nitrogen and sulfur-containing secondary metabolite found primarily in Brassicaceae plants. Other secondary metabolites may include allelochemicals.

In some examples, PMPs are not typically produced by plants, but are generally associated with specific markers (eg, lipids, polypeptides) that are generally associated with other organisms (eg, markers of animal EV, bacterial EV or fungal EV). Alternatively, it can also be identified as being produced using a plant EV based on a deletion of the polynucleotide). For example, in some examples, PMP lacks the lipids typically found in animal EVs, bacterial EVs or fungal EVs. In some examples, PMP lacks lipids typical of animal EVs (eg, sphingomyelin). In some examples, PMP does not contain lipids typical of bacterial EVs or bacterial membranes (eg, LPS). In some examples, PMP lacks lipids typical of fungal membranes (eg, ergosterols).

Capable of identifying small molecules (eg, mass spectrometry, mass spectrometry), lipids (eg, mass spectrometry, mass spectrometry), proteins (eg, mass spectrometry, immunoblotting) or nucleic acids (eg, PCR analysis) Any technique known in the art can be used to identify plant EV markers. In some examples, the PMP compositions described herein contain a detectable amount, eg, a predetermined threshold amount, of the plant EV markers described herein.

C. Drug Loading PMPs can be modified to contain heterologous plant modifiers, such as pesticide agents or repellents such as those described herein. PMP is a variety of means that allow delivery of a drug to a target plant, such as encapsulation of the drug, incorporation of the component into the lipid bilayer structure, or binding of the surface to the component of the lipid bilayer structure of the PMP (eg,). , Conjugation) allows these agents to be carried or coupled to them.

Heterologous plant modifiers can be incorporated or loaded into or on the PMP by any method known in the art that allows direct or indirect binding of the PMP to the drug. Heterologous plant modifiers can be obtained by in vivo methods (eg, by production of PMP from implanters, eg, transgenic plants containing heterologous agents) or in vitro (eg, by tissue or cell culture) or by both in vivo and in vitro methods. It can be incorporated into PMP.

In an example of loading a heterologous plant modifier into a PMP in vivo, the PMP can be produced using an EV or a fragment or portion thereof or an extract containing the EV loaded with an implanter. The implanter method comprises the expression of a heterologous plant modifier in a plant genetically modified to express the heterologous plant modifier for loading into an EV. In some examples, the heterologous plant modifier is exogenous to the plant. Alternatively, heterologous plant modifiers can be found naturally in plants, but can be engineered to be expressed at levels higher than those found in non-genetically modified plants.

In some examples, PMP can be loaded in vitro. Without limitation, physical, chemical and / or biological methods (eg, in tissue or cell cultures) can be used to load the substance onto or into the PMP (eg, encapsulated by PMP). can do). For example, the heterologous plant modifier can be introduced into the PMP by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction or extrusion. Various methods, such as HPCL (eg, for small molecule evaluation); immunoblotting (eg, for protein evaluation); and quantitative PCR (eg, for nucleotide evaluation), to confirm the presence or level of loaded drug. For evaluation) can be used to evaluate the loaded PMP. However, it should be understood by those skilled in the art that the loading of the substance of interest into the PMP is not limited to the methods exemplified above.

In some examples, the heterologous plant modifier can be conjugated to the PMP, in which case the heterologous plant modifier is indirectly or directly bound or linked to the PMP. For example, one or more plant modifiers are chemically linked to the PMP so that the one or more plant modifiers are directly linked to the lipid bilayer of the PMP (eg, by covalent or ionic bond). be able to. In some examples, the conjugate of various plant modifiers to PMP first combines one or more heterologous plant modifiers in a suitable solvent with a suitable cross-linking agent (eg, N-ethylcarbo-diimide ("EDC"). "); This is generally used as a carboxyl activator for the amide bond with the primary amine and can also be achieved by mixing with a phosphate group). After sufficient incubation time to allow binding of the cross-linking agent and the cross-linking agent, the cross-linking agent / cross-linking agent mixture is combined with PMP and after a further incubation time, the sucrose gradient (eg, 8, 30, etc.). 45 and 60% sucrose gradients) can be used to separate free heterologous plant modifiers and free PMPs from PMP-conjugated plant modifiers. PMP conjugated to the plant modifier as part of the step of matching the mixture to the sucrose gradient and the accompanying centrifugation step is now recognized as a band in the sucrose gradient, and thus the conjugated PMP is then added. It can be collected, washed and dissolved in a solution suitable for use as described herein.

In some examples, for example, before and after delivery of PMP to a plant, PMP is stably bound to a heterologous plant modifier. In another example, the PMP is bound to the heterologous plant modifier such that, for example, after delivery of the PMP to the plant, the heterologous plant modifier is dissociated from the PMP.

The PMP can also be further modified with other elements (eg, lipids such as sterols such as cholesterol; or small molecules) to further modify the functional and structural characteristics of the PMP. For example, the PMP can be further modified with a stabilizing molecule that enhances the stability of the PMP (eg, stable at room temperature for at least 1 day and / or at 4 ° C. for at least 1 week).

The PMP can be loaded with different concentrations of heterologous plant modifiers, depending on the particular agent or use. For example, in some examples, the plant modification compositions disclosed herein are about 0.001, 0.01, 0.1, 1.0, 2, 3, 4, 5, 6, 7, ... To contain more than 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 95 (or any range from about 0.001 to 95) or more wt% plant modifiers. Then load it into PMP. In some examples, the plant modification composition is about 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 , 1.0, 0.1, 0.01, 0.001 (or any range from about 95 to 0.001) or less wt% plant modifiers are loaded into the PMP. For example, the plant modification composition is about 0.001 to about 0.01 wt%, about 0.01 to about 0.1 wt%, about 0.1 to about 1 wt%, about 1 to about 5 wt% or about 5 to about. It may contain 10 wt%, about 10 to about 20 wt% plant modifiers. In some examples, PMP is a plant of about 1, 5, 10, 50, 200 or 500, 1,000, 2,000 (or any range from about 1 to 2,000) or more μg / ml. Modifiers can be loaded. In some examples, μg / ml plant modifications of about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range from about 2,000 to 1) or less. The agent can be loaded into the PMP.

In some examples, the PMP contains at least 0.001 wt%, at least 0.01 wt%, at least 0.1 wt%, at least 1.0 wt%, at least 2 wt% of the plant modification compositions disclosed herein. At least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least Load 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt% or at least 95 wt% plant modifier. In some examples, PMP is at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 200 μg / ml, at least 500 μg / ml, at least 1,000 μg / ml. A plant modifier can be loaded in ml, at least 2,000 μg / ml.

Examples of specific plant modifiers that can be loaded into PMP are further outlined in the section entitled "Heterologous Plant Modifiers".

D. Formulations The plant-modified composition can be formulated with other substances such as agriculturally acceptable carriers to facilitate application, handling, transport, storage and activity. Plant modification compositions include, for example, baits, concentrated emulsions, powders, emulsions, fumigants, gels, granules, microcapsule encapsulants, seed treatment agents, suspension concentrates, suspo emulsions, tablets, water-soluble liquids, water. It can be formulated into dispersible granules or dry flowable agents, hydrated powders and trace solutions.

The plant modification composition can be applied as an aqueous suspension or emulsion prepared from a concentrated formulation of such substances. Such water-soluble, water-suspendable or emulsifying formulations can be either solids (usually known as wettable powders) or water-dispersible granules, or liquids usually known as emulsions, or aqueous suspensions. obtain. Wettable powders that can be compressed to form water-dispersible granules include a homogeneous mixture of plant-modified compositions, carriers and surfactants. The carrier is usually selected from attapulsite clay, montmorillonite clay, diatomaceous earth or purified silicic acid. Effective surfactants containing from about 0.5% to about 10% wettable powder include nonionic surfactants such as sulfonated lignin, concentrated naphthalene sulfonate, naphthalene sulfonate, alkylbenzene sulfonate, alkyl sulfonate and ethylene oxide adducts of alkylphenol. Examples include ionic surfactants.

The emulsion contains a suitable concentration of PMP and / or plant modifier, such as about 50-about 500 grams per liter of liquid dissolved in a carrier, which is either a water-soluble solvent or a mixture of a water-soluble organic solvent and an emulsifier. Can be. Useful organic solvents include aromatic oils, especially xylene and petroleum fractions, especially high boiling naphthalene and olefin moieties of petroleum, such as heavy aromatic naphtha. In addition, other organic solvents such as terpene solvents containing rosin derivatives, aliphatic ketones such as cyclohexanone and complex alcohols such as 2-ethoxyethanol can be used. Suitable emulsifiers for emulsions are selected from common anionic and nonionic surfactants.

Aqueous suspensions include suspensions of water-insoluble pesticides dispersed in an aqueous carrier with a plant modification composition ranging from about 5% to about 50% by weight. Suspensions are prepared by finely grinding the active material and mixing it vigorously in a carrier containing water and a surfactant. Also, materials such as inorganic salts and synthetic or natural rubber may be added to increase the density and viscosity of the aqueous carrier.

The plant modification composition can be applied as a granular composition, which is particularly useful for soil applications. The granular composition usually contains from about 0.5% to about 10% by weight of the plant modification composition dispersed on a carrier containing clay or a similar substance. Such compositions are usually prepared by dissolving the formulation in a suitable solvent and then applying to a preformed granular carrier with a suitable particle size in the range of about 0.5 to about 3 mm. Such compositions can be formulated by making a duff or paste of the carrier and compound, grinding and then drying to obtain the desired granular size.

The powder containing the present composition is prepared by homogeneously mixing the powdered plant modification composition with a suitable powdered carrier such as kaolin clay, ground volcanic rock and the like. The powder may preferably occupy about 1% to about 10% of the packet. The powder can be applied as a seed powder coat or leaf application on a dust blower machine.

Similarly, it is also practical to apply the present formulation in the form of a suitable organic solvent, usually a solution in petroleum, for example spray oil, which is widely used in agricultural chemistry.

The plant modification composition can also be applied in the form of an aerosol composition. In such compositions, the packet is dissolved or dispersed in a carrier that is a pressure generating spray mixture. The aerosol composition is packaged in a container and the mixture is administered from this container via a atomizing valve.

Another embodiment is an oil-in-water emulsion, where the emulsion comprises oil droplets, each of which comprises a lamellar liquid crystal coating and is dispersed in an aqueous phase, wherein each oil droplet is: Contains at least one agriculturally effective compound, (1) at least one nonionic lipophilic surfactant, (2) at least one nonionic hydrophilic surfactant, and (3) at least one ionic surfactant. Individually coated with a monolamellar or oligolamellar layer containing the agent, in this case the oil droplets have an average particle size of less than 800 nanometers. More detailed information about the embodiment is disclosed in US Patent Application Publication No. 2007070734 published February 1, 2007. For ease of use, this embodiment is referred to as "OIWE".

In addition, in general, when the molecules disclosed above are used in a formulation, such formulation can also contain other components. These ingredients include, but are not limited to, wetting agents, spreading agents, adhesives, penetrants, buffers, sequestrants, drift control additives (which are non-exclusive and non-mutually exclusive lists). , Compatible agents, antifoaming agents, detergents and emulsifiers. Some ingredients are listed below.

A wetting agent, when added to a liquid, is a substance that enhances the spreading or penetrating power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spread. Wetting agents have two main functions of agricultural chemicals: during the treatment and manufacturing process, the rate of wetting of the powder with water is increased to produce concentrates or suspensions for soluble liquids; water in a spray tank. And during the product mixing process, it is used to shorten the wetting time of the wettable powder and improve the penetration of water into the water-dispersible granules. Examples of wetting agents used in wettable powders, suspensions and water-dispersible granule formulations are sodium lauryl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

The dispersant is a substance that adsorbs to the surface of the particles to promote the preservation of the dispersed state of the particles and prevent the particles from reaggregating. Dispersants are added to the pesticide formulation to facilitate dispersion and suspension during the manufacturing process and to ensure that the particles are redispersed in the water in the spray tank. Dispersants are widely used in wettable powders, suspensions and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb to the particle surface, resulting in charge or steric hindrance to particle reaggregation. The most commonly used surfactants are anions, nonionics or mixtures of the two. For wettable powders, the most common dispersant is sodium lignosulfonate. For suspending agents, polyelectrolytes such as sodium naphthalene sulfonic acid formaldehyde condensates are used to achieve very good adsorption and stabilization. Tristyrylphenol ethoxylate phosphate ester is also used. Nonionic surfactants such as alkylarylethylene oxide condensates and EO-PO block copolymers may be combined with anionic surfactants as suspension concentrates. In recent years, a new type of very high molecular weight polymer surfactant has been developed as a dispersant. They have a very long hydrophobic "skeleton" and numerous ethylene oxide chains that form the "teeth" of the "comb" surfactant. These high molecular weight polymers can impart very good long-term stability to the suspending agent because the hydrophobic skeleton has many fixation points on the particle surface. Examples of dispersants used in pesticide formulations are sodium lignosulfonate; sodium naphthalene sulfonic acid formaldehyde condensate; tristyrylphenol ethoxylate phosphate ester; aliphatic alcohol ethoxylates; alkyl ethoxylates; EO-PO (ethylene oxide- Propropylene oxide) block copolymers; and graft copolymers.

An emulsifier is a substance that stabilizes the suspension of droplets in one liquid phase in another liquid phase. Without the emulsifier, the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain an alkylphenol or fatty alcohol with 12 or more ethylene oxide units and an oil-soluble calcium salt of dodecylbenzene sulfonic acid. A range of hydrophilic-lipophilic balance (“HLB”) values from 8 to 18 usually provides excellent stable emulsions. Emulsion stability can sometimes be improved by the addition of small amounts of EO-PO block copolymer surfactants.

The solubilizer is a surfactant that forms an aqueous micelle solution having a concentration exceeding the critical micelle concentration. Therefore, micelles can dissolve or solubilize water-insoluble materials within the hydrophobic portion of micelles. The types of surfactants commonly used for solubilization are nonionic surfactants, sorbitan monooleates, sorbitan monooleate ethoxylates and oleic acid methyl esters.

When the surfactant is used alone or in combination with other additives such as mineral oil or vegetable oil as an adjunct to the spray tank mix to improve the biological performance of the plant modification composition to the target. There is also. The type of surfactant used for bioenhancement generally varies depending on the nature and mode of action of the plant modification composition. However, these are often nonionic surfactants such as alkyl ethoxylates; linear aliphatic alcohol ethoxylates; aliphatic amine ethoxylates.

The carrier or diluent in the pesticide formulation is a material added to the plant modification composition to provide the formulation of the required strength. The carrier is usually a material with high absorbency, whereas the diluent is usually a material with low absorbency. The carrier and diluent are used in the preparation of powders, wettable powders, granules and water-dispersible granules.

The organic solvent is mainly used for emulsions, oil-in-water emulsions, suspo emulsions and very small amounts, and to a lesser extent, granules. A mixture of solvents may also be used. The first major group of solvents are aliphatic paraffin oils such as kerosene or purified paraffin. The second major group (and most commonly) contains aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. The chlorinated hydrocarbon is useful as a co-solvent to prevent crystallization of the plant modification composition when emulsifying the formulation into water. Alcohol may be used as a co-solvent to enhance the dissolving power. Other solvents may include vegetable oils, seed oils and esters of plants and seed oils.

Thickeners or gelling agents are formulations of suspensions, emulsions and saspo emulsions primarily to alter the rheology or fluidity of the liquid and to prevent the separation and precipitation of dispersed particles or droplets. Used for. Thickeners, gelling agents and anti-precipitation agents generally belong to two categories: water-insoluble particles and water-soluble polymers. It is possible to produce a suspension product using clay and silica. Examples of these types of materials include, but are not limited to, montmorillonite clay, bentonite, magnesium aluminum silicate and attapurgate. Water-soluble polysaccharides have been used for many years as thickening-gelling agents. The most commonly used types of polysaccharides are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum; locust bean gum; carrageenan; alginate; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). Other types of anti-precipitation agents are based on modified starch, polyacrylates, polyvinyl alcohol and polyethylene oxide. Another excellent anti-precipitation agent is xanthan gum.

Microorganisms can cause spoilage of formulated products. Therefore, preservatives are used to eliminate or suppress the action of microorganisms. Examples of such agents include, but are not limited to: propionic acid and its salts; sorbic acid and its sodium or potassium salts; benzoic acid and its salts; p-hydroxybenzoic acid sodium salts; p-hydroxybenzoic acid. Methyl acidate; as well as 1,2-benzisothiazolin-3-one (BIT).

The presence of surfactant often foams the aqueous formulation during the mixing process in the production and application from the spray tank. Defoamers are often added during the production phase or before bottling to reduce the tendency to foam. Generally, there are two types of antifoaming agents, namely silicone and non-silicone. Silicone is usually an aqueous emulsion of dimethylpolysiloxane, and the non-silicone defoaming agent is a water-insoluble oil such as octanol and nonanol or silica. In either case, the function of the defoaming agent is to transfer the surfactant from the air-water interface.

"Green agents" (eg, auxiliaries, surfactants, solvents) can reduce the overall environmental footprint of crop protection formulations. Greening agents are biodegradable and generally come from natural and / or sustainable sources such as plant and animal sources. Specific examples are vegetable oils, seed oils and esters thereof, as well as alkoxylated alkylphenol polyglucosides.

In some examples, the plant modification composition can be freeze-dried or lyophilized. See U.S. Pat. No. 4,311,712. The plant modification composition can later be reconstituted by contact with water or another liquid. Other ingredients, such as other plant modifiers, pesticide agents, agriculturally acceptable carriers or other substances according to the formulations described herein can be added to the lyophilized or reconstituted product.

Other optional features of the composition include a carrier or delivery vehicle that protects the plant modification composition from UV and / or acidic conditions. In some examples, the delivery vehicle contains a pH buffer. In some examples, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, eg, about 5.0 to about 8.0, about 6.5 to about 7. Includes one pH range of 5.5 or about 6.5 to about 7.0.

For more information on pesticide formulations, see: “Chemistry and Technology of Agrochemical Formulas” edited by D.I. A. Knowles, copyright 1998 by Klewer Academic Publishers. See also: “Insectides in Agriculture and Insecticide and Prospects” by A. et al. S. Perry, I. Yamamoto, I. Ishayaya, and R. Perry, copyright 1998 by Springer-Verlag.

II. Plant Modification Methods The plant modification compositions described herein are useful in a variety of methods. The present invention comprises the step of delivering the plant modification composition described herein to a plant such as the plant described herein. The composition and related methods can be used to modify a plant or plant part (eg, leaves, roots, flowers, fruits or seeds) to increase the adaptability of the plant. Details of each of these methods are described in more detail below.

A. Delivery to Plants herein provides a method of delivering a plant modification composition to a plant (eg, the plant disclosed in the section entitled "Plants"). Included are methods for delivering a plant modification composition to a plant by contacting the plant or a portion thereof with the plant modification composition. This method is useful for modifying plants to increase their fitness.

In one aspect, herein is a method of increasing the adaptability of a plant in order to increase the adaptability of the plant as compared to an untreated plant (a plant to which the plant modification composition has not been delivered). Provided are methods comprising delivering the plant modification compositions described herein to a plant (eg, in an effective amount and duration).

The increase in plant adaptability as a result of delivery of the plant modification composition can manifest itself in several ways, for example thereby good production of the plant, eg improved yield, plant vitality or harvested from the plant. Improving product quality, improving pre-harvest or post-harvest traits that may be desirable for agriculture or gardening (eg, taste, appearance, shelf life) or otherwise beneficial to humans (eg, improving traits) , Reduced production of allergens). The improved yield of the plant is delivered under the same conditions, but compared to the yield of the same product of the plant produced without the application of the composition or without the conventional plant modifier (eg, PMP free). Yields of plant products (eg, measured by plant biomass, cereals, seeds or fruit yields, protein content, carbohydrate or oil content or leaf area) in measurable amounts compared to the application of plant modifiers. Regarding the increase in. For example, the yields are at least about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50. %, About 60%, about 70%, about 80%, about 90%, about 100% or more than 100% can be increased. In some examples, the method increases yields by about 2-fold, about 5-fold, about 10-fold, about 25-fold, about 50-fold, about 75-fold, 100-fold or more than 100-fold with respect to untreated plants. Is effective for. Yield can be expressed in terms of the amount per weight or volume of the plant or plant product on some basis. Criteria can be expressed in terms of time, acreage, weight of plants produced or amount of raw materials used. For example, such methods can increase the yield of plant tissues including, but not limited to, seeds, juices, grains, fruits, tubers, roots and leaves.

The increase in plant adaptability as a result of delivery of the plant modification composition is under the same conditions, but without administration of the composition or delivery with conventional plant modification agents (eg, without PMP). Increase or improvement of the following items by measurable or recognizable amount compared to the same factors of plants produced with phytomodifiers): Vitality assessment, Crop (number of plants per unit area) ), Plant height, stalk circumference, stalk length, number of leaves, leaf size, plant canopy, appearance (more green leaf color, etc.), root evaluation, sprouting, protein content, increased swelling, greater Leaves, more leaves, less dead rooted leaves, stronger vegetation power, less fertilizer required, less seeds needed, more productive vegetation, earlier flowering, earlier It can also be measured by other means such as grain or seed maturity, less plant lodging (yrowning), increased sprout growth, early germination or any combination thereof.

Accordingly, herein is a method of modifying a plant comprising modifying the plant to deliver an effective amount of any of the plant modification compositions provided herein. Introduces beneficial traits in plants or against untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70). %, 80%, 90%, 100% or more than 100%) methods are provided. In particular, this method provides plant fitness to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%). , 80%, 90%, 100% or more than 100%).

In some cases, increased adaptability of plants is disease resistance, drought resistance, heat resistance, cold resistance, salt resistance, metal resistance, herbicide resistance, drug resistance, water utilization efficiency, nitrogen utilization, nitrogen stress resistance, Nitrogen fixation, pest resistance, herbicide resistance, pathogen resistance, yield, yield under water shortage conditions, vitality, growth, photosynthetic ability, nutrition, protein content, carbohydrate content, oil content, biomass, sprout length, root length, roots Structure, seed weight or amount of harvestable product (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% increase.

In some examples, increased fitness is in development, growth, yield, resistance to abiotic stressors or resistance to biological stressors (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%) increase. Abiotic stress refers to environmental stress conditions to which a plant or plant part is exposed, including, for example, drought stress, salt stress, heat stress, cold stress and malnutrition stress. Biological stress refers to environmental stress conditions to which a plant or plant part is exposed, including, for example, nematode stress, herbivorous insect stress, fungal pathogen stress, pathogen stress or viral pathogen stress. Stress can be temporary, eg, hours, days, months, or permanent throughout the life of the plant.

In some examples, increased fitness of the plant is in the quality of the product harvested from the plant (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%). , 60%, 70%, 80%, 90%, 100% or more than 100%) improvement. For example, an increase in the fitness of a plant can be an improvement in the commercially favorable characteristics (eg, taste or appearance) of the product harvested from the plant. In another example, the increase in fitness of a plant is during the storage period of the product harvested from the plant (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%). , 60%, 70%, 80%, 90%, 100% or more than 100%) increase.

Instead, increased fitness can be a change in traits that is beneficial to human or animal health, such as reduced production of allergens. For example, increased fitness is (eg, about 1%, 2%, 5%, 10%, 20%, 30%) in the production of allergens (eg, pollen) that stimulate an immune response in animals (eg, humans). , 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%).

Plant modifications (eg, increased fitness) can result from modifications of one or more plant parts. For example, plants can be modified by contact with plant leaves, seeds, pollen, roots, fruits, shoots, flowers, cells, protoplasts or tissues (eg, meristem). Accordingly, in another embodiment, herein, a method of increasing fitness of a plant, the pollen of the plant is contacted with an effective amount of any of the plant modification compositions provided herein. Plant fitness to untreated plants, including steps (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80). %, 90%, 100% or more than 100%) methods are provided.

In yet another embodiment, herein, a method of increasing the fitness of a plant, the step of contacting the seeds of a plant with an effective amount of any of the plant modification compositions provided herein. Adaptation of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%). , 90%, 100% or more than 100%).

In yet another embodiment, the present specification comprises contacting a plant protoplast with an effective amount of any of the plant modification compositions provided herein, comprising adapting the plant to an untreated plant. Increased degree (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or over 100%) A way to make it is provided.

In another embodiment, herein, a method of increasing fitness of a plant, the step of contacting a plant cell of a plant with an effective amount of any of the plant modification compositions provided herein. Adaptation of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%). , 90%, 100% or more than 100%).

In yet another embodiment, herein, a method of increasing the fitness of a plant, the meristem of the plant is contacted with an effective amount of any of the plant modification compositions provided herein. Plant fitness to untreated plants, including steps (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80). %, 90%, 100% or more than 100%) methods are provided.

In yet another embodiment, the present specification comprises a method of increasing the fitness of a plant, comprising contacting the plant germ with an effective amount of the plant modification composition provided herein. Fitness of plants to untreated plants (eg, about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) , 100% or more than 100%) are provided.

B. Methods of Application The plants described herein can be exposed to any of the compositions described herein in any suitable manner that allows delivery or administration of the composition to the plant. The plant modification composition can be delivered either alone or in combination with other active or inactive substances (eg, fertilizers), eg, concentrates formulated to deliver an effective concentration of the plant modification composition. It can be applied in the form of liquids, gels, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, for example by spraying, injection (microinjection), via plants, injection, immersion. The amounts and sites of application of the compositions described herein are generally plant habits, sites of life cycle where plants can be targeted by plant-modified compositions, and sites to which they are applied. Determined by the physical and functional properties of the plant modification composition.

In some examples, the composition is sprayed directly onto a plant, eg, a crop, for example by backpack spraying, aerial spraying, crop spraying / dusting, and the like. In the example where the plant modification composition is delivered to the plant, the plant to which the plant modification composition is administered can be at any stage of plant growth. For example, the formulated plant modification composition can be applied as a seed coating or rooting treatment early in plant growth or as a whole plant treatment late in the crop cycle. In some examples, the plant modification composition may be applied to the plant as a topical agent.

In addition, the plant modification composition can be applied as a permeable agent that is absorbed and distributed through the tissues of the plant or pest (eg, in the soil in which the plant grows or in the water used to water the plant). In some examples, the plant or feed organism can be genetically transformed to express a plant modification composition.

Delayed or continuous release also allows the coating to dissolve or decompose in the environment of use by coating the plant-modified composition or the composition containing the plant-modified composition with a soluble or biodegradable coating layer such as gelatin. It can then be achieved by making the plant modification composition available, or by dispersing the agent in a soluble or degradable matrix. Advantageously, such continuous release and / or dispensing means devices can be used to maintain one or more effective concentrations of the plant modification compositions described herein at all times.

In some examples, the plant modification composition is delivered to parts of the plant, such as leaves, seeds, pollen, roots, fruits, shoots or flowers or their tissues, cells or protoplasts. In some examples, the plant modification composition is delivered to plant cells. In some examples, the plant modification composition is delivered to the plant protoplasts. In some examples, the plant modification composition is delivered to the tissue of the plant. For example, the composition can be delivered to a plant meristem (eg, apical meristem, lateral meristem or internode meristem). In some examples, the composition may be delivered to plant permanent tissue (eg, single tissue (eg, parenchyma, collenchyma or thick membrane tissue) or complex permanent tissue (eg, xylem or phloem). In some examples, the composition is delivered to the plant germ.

In some examples, the plant modification composition, for field application, is the amount of PMP per hectare (g / ha or kg / ha) or the active ingredient per hectare (eg, plant modifier) or acid equivalent. It can be recommended as the amount of material (kg ai / ha or ai / ha). In some examples, in order to achieve the same results as when a PMP-free plant modifier was applied to the composition, a smaller amount of the plant modifier was added to the soil, plant medium, seeds in the composition. It may need to be applied to plant tissues or plants. For example, the amount of plant modifier is about 2, 3, 4, 5, 6, 7, 8 compared to direct application of the same plant modifier applied in a non-PMP composition, eg the same plant modifier without PMP. , 9, 10, 15, 20, 30, 50 or 100 times (or any range from about 2 to about 100 times, eg about 2-10 times; about 5-15 times, about 10-20 times; about 10 times. ~ 50 times) can be applied at lower levels. The plant modification compositions of the present invention are in various amounts per hectare, eg, about 0.0001, 0.001, 0.005, 0.01, 0.1, 1, 2, 10, 100, 1, It can be applied at 000, 2,000, 5,000 (or any range of about 0.0001 to 5,000) kg / ha. For example, it is about 0.0001 to about 0.01, about 0.01 to about 10, about 10 to about 1,000, and about 1,000 to about 5,000 kg / ha.

III. Plants A variety of plants can be delivered with or treated with the plant modification compositions described herein. Plants to which the plant modification composition can be delivered (ie, "processed") according to the method include whole plants and parts thereof, such as, but not limited to, sprout plant organs / structures (eg, leaves, etc.). Stems and stalks), roots, flowers and flower vessels / structures (eg, follicles, stalks, petals, buds, carpels, anthers and ovules), seeds (including embryos, endosperms, cotyledons and seed coats) and fruits (mature offspring). Tufts), plant tissues (eg, vascular tissue, basic tissue, etc.) and cells (eg, guard cells, egg cells, etc.) and their progeny. Plant parts are also sprouts, roots, stems, seeds, leaves, leaves, petals, flowers, germs, foliage, branches, peduncles, internodes, bark, soft hair, splits, rhizomes, fronds, leaves. Plant parts such as flesh, pollen, and rooster can also be shown.

The types of plants that can be treated by the methods disclosed herein include higher and lower plant types, such as angiosperms (single-leaf and dicotyledonous plants), angiosperms, ferns, and toxas. , Pterophytes, lycophytes, mosses and algae (eg, multi-cell or single-cell algae). Plants that can be treated according to this method further include, but are not limited to, any tubule plant, such as monocotyledonous or dicotyledonous or numerous plants, such as, but not limited to, alfalfa, apple, rapepsis, banana, barley. , Canola, Tougoma, Kiku, Clover, Cacao, Coffee, Cotton, Cottonseed, Corn, Cranbe, Cranberry, Cucumber, Dendrobium, Yamaimo, Eucalyptus, Uchinokegusa, Flax, Graradius, Liaceae, Flaxseed, Millon, Mask Melon, Karashi , Otsumugi, oilseed rape, oilseed rape, papaya, peanuts, pineapple, ornamental plants, green beans, potatoes, rapeseed, rice, limewood, ryegrass, benibana, sesame, morokoshi, soybeans, tensai, sugar cane, sunflower, strawberries, tobacco, tomatoes, turfgrass, Wheat and vegetable crops such as lettuce, celery, broccoli, cauliflower, rapeseed; fruits and fruit trees such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; grapes (eg, grapes) (Vegetable garden), grapes such as kiwi, hops; fruit shrubs such as raspberries, blackberries, rapeseed and strawberry; forest trees such as oilseed rape, pine, fir, maple, oak, walnuts, poplar; and even alfalfa, canola, Examples include sesame seeds, corn, cotton, crumbets, flax, flaxseed, rapeseed, oilseed rape, oilseed rape, peanuts, potatoes, rice, benibana, sesame seeds, soybeans, tensai, sunflowers, tobacco, tomatoes and wheat. The plants that can be processed according to the method of the present invention include all crops, such as forage crops, oily seed crops, grain crops, fruit crops, vegetable crops, textile crops, spice crops, fruit crops, turf crops, sugar crops, beverages. Examples include crops and forest crops. In a particular example, the crop plant treated by this method is a soybean plant. In another particular example, the crop plant is wheat. In a particular example, the crop plant is corn. In a particular example, the crop plant is cotton. In a particular example, the crop plant is alfalfa. In a particular example, the crop plant is sugar beet. In a particular example, the crop plant is rice. In a particular example, the crop plant is a potato. In a particular example, the crop plant is tomato.

In a particular example, the plant is a cereal. Examples of such cereal plants include, but are not limited to, monocotyledonous or dicotyledonous plants, such as, but not limited to, allium spp., Allium spp. Amaranthus spp., Pineapple (Ananas comosos), Celery (Apium plants), Aracchys spp, Asparagus (Asparagus officeis), Beet (Beta vulgas) sp. Brassica napus, Brassica rapa ssp. (Canola, rapeseed, turnip rape), Camellia sinensis, Camellia sinensis, Canna indica na , Allium spp., Castanea spp., Endive (Cichorium endiva), Citrulus lanatus, Citrus spp., Cocos spp. , Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucurbita spp. Cucumis spp.), Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Allium spp., Allium spp., Allium spp., Allium spp., Allium spp., Allium spp. (Glycine spp.) (For example, Glycine max, Soya hispida or Soya max), Gossypium hirsutum, Helianthus sp. .. (Eg Helianthus annuus), Hibiscus spp., Hordeum spp. (Eg, Hordeum vulgare), Ipomea batas (Ipomea ata) Juglans spp., Lactuca sativa, Linum sitatissimum, Litchi chinensis, Lotus spp, lotus spp. Lupinus spp., Lycopersicon spp. (For example, Lycopersicon esculenturn, Lycopersicon lycopercicum, Lycopersicon lycoper) spp.), Medicago sativa, Menta spp., Miscanthus sinensis, Morus nigra, Musa spp., Nikothiana sp. (Nicotiana spp.), Olea spp., Oryza spp. (For example, Oryza sativa, Oryza latifolia), Panicum milicaum. , Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Phaseolus spp., Pinus spip. , Pisum spp., Poa spp., Poa spp. Populus spp. ), Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rabarbarum sp. .), Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secare sp. (Secale cereal), Sesamum spp., Synapis spp., Solanum spp. (For example, Solanum tuberosum, Solanam intefolium). Solanum integrifolia or Solanum lycopersicum), Sorghum bicolor, Sorghum halepens, Sorghum halepense, Spinasia sp. (Theobroma cacao), Trifolium spp., Triticosekel lymphui, Triticum spp. (For example, Triticum estibum, Triticum astimum) Triticum turgidam, Triticum hybernum, Triticum macha, Triticum sativum, Triticum sativum, Triticum vulgare, Triticum vulgare, Triticum vulgare Vicia spp., Vigna spp., Viola sp. Viola odorata, Vitis spp. ) And feeds or legumes selected from Zea mays, ornamental plants, edible crops, trees or shrubs. In certain embodiments, the crop plant is rice, rape, canola, soybean, corn (corn), cotton, sugar cane, alfalfa, great millet or wheat.

In certain examples, the compositions and methods can be used to treat post-harvest plants or plant parts or feed products. In some examples, the food or feed product is a non-vegetable food or feed product (eg, a product edible by humans, animals or livestock (eg, mushrooms)).

The plants or parts used in the present invention include plants at any stage of plant development. In certain examples, delivery is at the stages of germination, sapling growth, vegetative growth and reproductive growth. In certain cases, delivery to the plant can be done at the vegetative and reproductive growth stages. In some examples, the composition is delivered to the pollen of the plant. In some examples, the composition is delivered to the seeds of the plant. In some examples, the composition is delivered to a plant protoplast. In some examples, the composition is delivered to plant tissue. For example, the composition can be delivered to a plant meristem (eg, apical meristem, lateral meristem or internode meristem). In some examples, the composition may be delivered to plant permanent tissue (eg, single tissue (eg, parenchyma, collenchyma or thick membrane tissue) or complex permanent tissue (eg, xylem or phloem). In some examples, the composition is delivered to the plant germ. In some examples, the composition is delivered to the plant cells. The stages of vegetative growth and reproductive growth are "adult" herein. Also called a "mature" plant.

In an example where the composition is delivered to a plant portion, the plant portion can be modified by a plant modifier. Alternatively, the plant modifier can be distributed to other parts of the plant and subsequently modified by the plant modifier.

IV. Heterogeneous plant modifiers Examples of the plant modifiers described herein include one or more heterologous plant modifiers. For example, PMP may encapsulate a heterologous plant modifier. Alternatively or additionally, the heterologous plant modifier may be embedded or conjugated to the surface of the PMP.

In some examples, the plant modifier may include peptides or nucleic acids. The plant modifier can be an agent that increases the fitness of various plants or an agent that targets one or more specific plants (eg, a specific species or genus of plants). In addition, in some examples, the plant-modifying composition comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) various plant-modifying agents. including. In some examples, the heterogeneous plant modifier comprises a ribonucleoprotein (RNP), eg, one or more RNA molecules associated with one or more RNA binding proteins.

In addition, in some examples, heterologous plant modifiers (eg, drugs containing nucleic acid molecules or peptides) can be modified. For example, the modification can be a chemical modification, eg, a conjugate to a marker, eg, a fluorescent marker or a radioactive marker. In another example, the modification is a conjugate or functional communication with a drug such as a lipid, glycan, polymer (eg, PEG), a moiety that enhances the stability, delivery, targeting, bioavailability or half-life of the cationic moiety. Can include.

Examples of heterologous plant modifiers (eg, peptides or nucleic acids) that can be used in the plant modification compositions and methods disclosed herein are outlined below.

A. Polypeptides The plant modification compositions described herein (eg, PMPs) may comprise heterologous polypeptides. In some examples, the plant modification compositions described herein include a polypeptide or a functional fragment or derivative thereof that modifies a plant (eg, increases the fitness of the plant). For example, polypeptides can increase the fitness of plants. Plant modification compositions comprising the polypeptides described herein achieve (a) target levels (eg, predetermined or threshold levels) of polypeptide concentrations; and (b) modify plants (eg, plants). It can be contacted with the plant in sufficient quantity and time to increase fitness).

Examples of polypeptides that can be used in the present invention include enzymes (eg, metabolic recombinases, helicases, integrases, RNAse, DNAse or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell membrane permeation peptides, transcription factors, etc. Receptors, antibodies, nanobodies, gene editing proteins (eg, CRISPR-Cas system, TALEN or zinc fingers), riboproteins, protein aptamers or chaperones can be mentioned.

The polypeptides included in the present invention may include naturally occurring polypeptides or recombinantly produced variants. In some examples, the polypeptide can be a functional fragment or variant thereof (eg, a fragment or variant thereof that is active as an enzyme). For example, the polypeptide may be, for example, at least 70%, 71%, 72%, 73%, 74 with the sequence of the polypeptide described herein or a naturally occurring polypeptide over a specific region or over the entire sequence. %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, A functionally active variant of any of the polypeptides described herein having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Can be. In some examples, the polypeptide is at least 50% (eg, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more) identical to the protein of interest. Can have sex.

The polypeptides described herein can be incorporated into a composition for any of the uses described herein. The compositions disclosed herein are of any number or type (eg, class) of polypeptide, eg, at least about one polypeptide, 2, 3, 4, 5, 10, 15, 20 or more. It may contain a polypeptide. The suitable concentration of each polypeptide in the composition will vary depending on factors such as the efficacy and stability of the polypeptide, the number of individual polypeptides in the composition, the formulation and the method of application of the composition. In some examples, each polypeptide in the liquid composition is from about 0.1 ng / mL to about 100 mg / mL. In some examples, each polypeptide in the solid composition is from about 0.1 ng / g to about 100 mg / g.

Methods of making polypeptides are routine in the art. In general, Smales & James, Therapeutic Proteins (Eds.):; (. Eds) Methods and Protocols (Methods in Molecular Biology), Humana Press (2005) and Crommelin, Sindelar & Meibohm, Pharmaceutical Biotechnology: Fundamentals and Applications, the Springer (2013) Please refer.

Methods for making polypeptides include expression in plant cells, but recombinant proteins can also be made using insect cells, yeast, bacteria, mammalian cells or other cells under the control of appropriate promoters. can. Mammalian expression vectors include non-transcriptional elements such as origins of replication, suitable promoters and enhancers as well as other 5'or 3'flanking non-transcriptional sequences and 5'or 3'non-transcriptional sequences such as the required ribosome binding site, polyadenylation. It may include an origin of replication, a splice donor and an acceptor site, and a terminal sequence. DNA sequences derived from the SV40 viral genome, such as SV40 origins, early promoters, enhancers, splices and polyadenylation sites, can also be used to provide other genetic elements required for expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory 12 Ress. There is.

Various mammalian cell culture systems can be used to express and produce recombinant polypeptide agents. Examples of mammalian expression systems include CHO cells, COS cells, HeLA and BHK cell lines. Methods of host cell culture for the production of protein therapeutics are described, for example, in Zhou and Kantardjeff (Eds.), Mammalian Cell Cultures for Biochemicals Manufacturing (Advances in Biochemical Engineering). For purification of proteins, France, Protein Biotechnology: Isolation, Proteinization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methols). For the formulation of a protein therapeutic agent, Meyer (Ed.), Therapeutic Protein Drug Products: Practical Products to formalation in the Laboratory, Manufacturing, and ders.

In some examples, the plant modification composition comprises an antibody or an antigen-binding fragment thereof. For example, the agents described herein can be antibodies that block or enhance the activity and / or function of plant elements. Antibodies can act as antagonists or agonists of polypeptides (eg, enzymes or cell receptors) in plants. The production and use of antibodies against target antigens are known in the art. Examples include antibody manipulation, use of denatured oligonucleotides, 5'-RACE, phage display and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacokinetics; antibody purification and storage; and screening and labeling techniques. For a method for producing a recombinant antibody, refer to the following: Zhiqiang An (Ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1st Edition, Wiley, 2009, and even Greenbody (Greenfiel). Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 2013.

B. Nucleic Acids In some examples, the PMPs described herein include heterologous nucleic acids. Numerous nucleic acids are useful in the plant modification compositions and methods described herein. The plant modification compositions disclosed herein are any number or type (eg, class) of nucleic acid (eg, DNA or RNA molecule, eg, mRNA, guide RNA (gRNA) or inhibitory RNA molecule (eg, eg). siRNA, shRNA or miRNA) or hybrid DNA-RNA molecules), including, for example, nucleic acids of at least about 1 class or variant or nucleic acids of 2, 3, 4, 5, 10, 15, 20 or more classes or variants. obtain. The suitable concentration of each nucleic acid in the composition varies depending on factors such as the efficacy and stability of the nucleic acid, the number of individual nucleic acids, the formulation, the method of applying the composition, and the like. Examples of nucleic acids useful in the present invention include antisense RNA, dicer substrate small interfering RNA (disRNA), small interfering RNA (siRNA), small hairpin (shRNA), microRNA (miRNA), (asymmetric interfering RNA). ) AiRNA, Peptide Nucleic Acid (PNA), Morphorino, Locked Nucleic Acid (LNA), piwi-Interacting RNA (piRNA), Ribozyme, Deoxyribozyme (DNAzyme), Aptamar (DNA, RNA), Circular RNA (circRNA), Guide RNA ( Contains gRNA) or DNA molecules. The nucleic acid can be attached to other elements (eg, polypeptides) in any way known in the art to ensure that the nucleic acid is stable, eg, nuclease resistant. For example, nucleic acids can be nuclease resistant when complexed with proteins.

Plant modification compositions comprising the nucleic acids described herein achieve (a) target level (eg, predetermined or threshold level) nucleic acid concentrations; and (b) modify plants (eg, plant fitness). Can be contacted with the plant in sufficient quantity and time.

(A) Nucleic acid encoding a polypeptide In some examples, the plant modification composition comprises a nucleic acid encoding a polypeptide. Nucleic acids encoding polypeptides are about 10 to about 50,000 nucleotides (nt), about 25 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200 to about 300 nt, About 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 ~ 5,000 nt, about 5,000 to about 6,000 nt, about 6,000 to about 7,000 nt, about 7,000 to about 8,000 nt, about 8,000 to about 9000 nt, about 9000 to about 10,000 nt, about 10,000 to about 15,000 nt, about 10,000 ~ 20,000 nt, about 10,000 ~ about 25,000 nt, about 10,000 ~ about 30,000 nt, about 10,000 ~ about 40,000 nt, about 10,000 ~ about 45,000 nt, about 10,000 It can have a length of ~ about 50,000 nt or any range between them.

The plant modification composition may also contain a functionally active variant of the nucleic acid sequence of interest. In some examples, nucleic acid variants are, for example, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, with the sequence of nucleic acid of interest and over a designated region or over the entire sequence. 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% identity. In some examples, the invention comprises a functionally active polypeptide encoded by a nucleic acid variant as described herein. In some examples, functionally active polypeptides encoded by nucleic acid variants are, for example, at least 70% over a designated region or over the entire amino acid sequence with, for example, the sequence of the polypeptide of interest or a naturally occurring polypeptide sequence. , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Certain methods of expressing a nucleic acid encoding a protein may include expression in cells, including insects, yeasts, plants, bacteria or other cells, under the control of an appropriate promoter. Expression vectors include non-transcriptional elements such as origins of replication, suitable promoters and enhancers as well as other 5'or 3'flanking non-transcriptional sequences and 5'or 3'non-transcriptional sequences such as the required ribosome binding site, polyadenylation sites. , Splice donor and acceptor sites as well as termination sequences. DNA sequences derived from the SV40 viral genome, such as SV40 origins, early promoters, enhancers, splices and polyadenylation sites, can also be used to provide other genetic elements required for expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described in Green et al. , Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

Gene modification using a recombination method is known in the art. Nucleic acid sequences encoding the desired gene can be obtained using recombinant methods known in the art, for example, screening the library from cells expressing the gene, deriving the gene from a vector known to contain it. Alternatively, it can be obtained by direct isolation from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

Expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. Expression vectors may be suitable for replication and integration within the bacterium. Expression vectors may also be suitable for replication and expression in eukaryotes. A typical cloning vector contains a transcription and translation terminator, a promoter useful for expression of the starting sequence and the desired nucleic acid sequence.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located 30-110 base pairs (bp) upstream of the initiation site, but in recent years some promoters have been found to contain functional elements also downstream of the initiation site. The spacing between the promoter elements is often flexible, and the promoter function is preserved even if the elements are inverted or moved relative to each other. For thymidine kinase (tk) promoters, the spacing between promoter elements can be increased up to 50 bp until activity begins to diminish. Depending on the promoter, the individual elements may function either jointly or independently to activate transcription.

An example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked therein. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR). Promoters, MoMuLV promoters, trileukemia virus promoters, Epstein-Barr pre-virus promoters, Laus sarcoma virus promoters and human gene promoters such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters and creatin kinases. Promoters can be mentioned.

Instead, the promoter can be an inductive promoter. The use of an inducible promoter turns on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turns it off when expression is not required. Provides a molecular switch that can. Examples of inductive promoters include, but are not limited to, metallothionin promoters, glucocorticoid promoters, progesterone promoters and tetracycline promoters.

The expression vector to be introduced is either a selective marker gene or a reporter gene to facilitate the identification and selection of expressed cells from a population of cells to be transfected or infected via a viral vector. It may contain both. In other embodiments, selectivity markers may be carried on individual portions of DNA for use in co-transfection methods. Both selectivity markers and reporter genes can be flanked with appropriate regulatory sequences to allow expression in host cells. Useful selectivity markers include antibiotic resistance genes such as neo.

Reporter genes can be used to identify potentially transformed cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or is expressed by a recipient source and encodes a polypeptide whose expression is exhibited by some easily detectable property, such as enzymatic activity. Reporter gene expression is assayed at a suitable time point after DNA is introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or the green fluorescent protein gene (eg, Ui-Tei et al., FEBS Letters 479: 79-). 82,2000). Suitable expression systems are well known and can be prepared using known techniques or commercially available ones can be obtained. In general, constructs with a minimum of 5'flanking regions showing the highest levels of expression of the reporter gene are identified as promoters. These promoter regions can be linked to the reporter gene and can be used to evaluate the drug for its ability to regulate promoter-driven transcription.

In some examples, an organism can be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins can be modified for a particular time point, eg, the developmental or differentiated state of the organism. In one example, the invention comprises a composition for modifying the expression of one or more proteins, eg, a protein that affects the expression of activity, structure or function. The expression of one or more proteins can be restricted to a specific position or spread throughout the organism.

(B) Synthetic mRNA
The plant modification composition may include a synthetic mRNA molecule, eg, a synthetic mRNA molecule encoding a polypeptide. Synthetic mRNA molecules can be chemically modified, for example. The mRNA molecule can be synthesized, for example, chemically or transcribed in vitro. The mRNA molecule can be placed on a plasmid, such as a viral vector, bacterial vector or eukaryotic expression vector. In some examples, mRNA molecules can be delivered to cells by transfection, electroporation or transduction (eg, adenovirus or lentivirus transduction).

In some examples, the modified RNA agent of interest described herein has a modified nucleoside or nucleotide. Such modifications are known and are described, for example, in International Publication No. 2012/019168. Further modifications include, for example, International Publication No. 2015/038882 Pamphlet; No. 2015/038882 Pamphlet; No. 2015/089511 Pamphlet; No. 2015/196130 Pamphlet; No. 2015/196118 Pamphlet and No. It is described in the 2015/196128 A2 pamphlet.

In some examples, the modified RNA encoding the polypeptide of interest has one or more end modifications, such as a 5'cap structure and / or a poly A tail (eg, 100-200 nucleotides in length). The 5'cap structures are CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deraza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-. It can be selected from the group consisting of azido-guanosine. In some cases, the modified RNA contains a 5'UTR containing at least one Kozak sequence and a 3'UTR. Such modifications are known and are described, for example, in International Publication No. 2012/135805 and Pamphlet 2013/052523. Further end modifications are described, for example, in International Publication No. 2014/164253 and No. 2016/011306, International Publication No. 2012/045075 and No. 2014/03924. Chimeric enzymes for synthesizing cap RNA molecules (eg, modified mRNAs) that may contain at least one chemical modification are described in WO 2014/028429.

In some examples, modified mRNAs can be cyclized or concategorized to create translational competent molecules that assist in the interaction of polyA-binding proteins with 5'end-binding proteins. The mechanism of cyclization or concateration can occur through at least three different pathways: 1) chemistry, 2) enzymes, and 3) ribozyme catalysts. The newly formed 5'-/ 3'-bond can be either intramolecular or intermolecular. These modifications are described in International Publication No. 2013/151736 Pamphlet.

Methods of making and purifying modified RNA are known in the art. For example, modified RNA is made using in vitro transcription (IVT) enzyme synthesis only. Methods for making IVT polynucleotides are known in the art and are described in International Publication No. 2013/151666, No. 2013/151668, No. 2013/151663, No. 2013/151669, 2013/151670 pamphlet, 2013/151664 pamphlet, 2013/151665 pamphlet, 2013/151671 pamphlet, 2013/151672 pamphlet, 2013/151667 pamphlet and No. It is described in the 2013/151736 pamphlet. In the purification method, a surface linked to a plurality of timidines or derivatives thereof and / or a plurality of uracils or derivatives thereof (poly T / U) is brought into contact with a sample under conditions such that an RNA transcript binds to the surface. Then, a method of purifying an RNA transcript containing a poly A tail by eluting the purified RNA transcript from the surface (International Publication No. 2014/15231); a scalable method with a length of up to 10,000 nucleotides. Methods using ion (eg, anion) exchange chromatography that allow RNA separation (International Publication No. 2014/144767); and methods of subjecting modified mRNA samples to DNAse treatment (International Publication No. 2014/152030). including.

Formulations of modified RNA are known and are described, for example, in International Publication No. 2013/090648. For example, formulations include, but are not limited to, nanoparticles, polylactic / glycolic acid copolymers (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels. , Fibrin Hydrogel, Fibrin Lou, Fibrin Sealant, Fibrinogen, Trombin, Rapid Emission Lipid Nanoparticles (reLNP) and combinations thereof.

Modified RNAs encoding polypeptides in the fields of human diseases, antibodies, viruses and various in vivo environments are known, eg, WO 2013/151666, 2013/151668, 2013 /. Table 6 of the 151663 pamphlet, 2013/151669 pamphlet, 2013/151670 pamphlet, 2013/151664 pamphlet, 2013/151665 pamphlet, 2013/151736 pamphlet; Disclosed in Tables 6 and 7 of the 2013/151672 pamphlet; Tables 6, 178 and 179 of the 2013/151671 pamphlet; Tables 6, 185 and 186 of the 2013/151667 pamphlet. ing. Any of the aforementioned may be synthesized as an IVT polynucleotide, a chimeric polynucleotide or a cyclic polynucleotide, each containing one or more modified nucleotides or terminal modifications.

(C) Inhibiting RNA
In some examples, the plant modification composition comprises an inhibitory RNA molecule, eg, an inhibitory RNA molecule that acts via the RNA interference (RNAi) pathway. In some examples, the inhibitory RNA molecule reduces the level of gene expression in the plant and / or reduces the level of protein in the plant. In some examples, the inhibitory RNA molecule inhibits the expression of plant genes. For example, inhibitory RNA molecules can include small interfering RNAs, small hairpin RNAs and / or microRNAs that target one gene in a plant. Certain RNA molecules can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules typically contain RNA or RNA-like constructs containing 15-50 base pairs (eg, about 18-25 base pairs) and are identical (complementary) to the coding sequence in the expressed target gene in the cell. ) Or has a nearly identical (substantially complementary) nucleobase sequence. RNAi molecules include, but are not limited to, dicer substrate small interfering RNA (disRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), small hairpin RNA (SHRNA), and partially double-stranded (meroduplex). ), Dicer substrate and multivalent RNA interference (US Pat. Nos. 8,084,599, 8,349,809, 8,513,207 and 9,200, US Pat. 276 specification). shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene via RNAi. The shRNA can be delivered to cells in the form of a plasmid, eg, a viral or bacterial vector (eg, by transfection, electroporation or transduction). MicroRNAs are typically non-coding RNA molecules with a length of about 22 nucleotides. The miRNA suppresses mRNA by binding to a target site on the mRNA molecule and causing, for example, cleavage of the mRNA, destabilization of the mRNA or inhibition of translation of the mRNA. In some examples, the inhibitory RNA molecule reduces the level and / or activity of negative regulators of function. In another example, the inhibitory RNA molecule reduces the level and / or activity of a positive regulator of function. Inhibitor RNA molecules can be synthesized, for example, chemically or transcribed in vitro.

In some examples, the nucleic acid is DNA, RNA or PNA. In some examples, RNA is inhibitory RNA. In some examples, inhibitory RNA inhibits gene expression in plants. In some examples, nucleic acids are DNA molecules, pore-forming proteins, signaling that increase the expression of enzymes (eg, metabolic recombinases, helicases, integrases, RNAse, DNAse or ubiquitinated proteins) in mRNAs, modified mRNAs or plants. A ligand, a cell membrane permeabilizing peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene editing protein (eg, CRISPR-Cas system, TALEN or zinc finger), a riboprotein, a protein aptamer or a chapelon. In some examples, the nucleic acid is a DNA molecule, pore that increases the expression of an mRNA, modified mRNA or enzyme (eg, a metabolic enzyme, recombinase enzyme, helicase enzyme, integrase enzyme, RNAse enzyme, DNAse enzyme or ubiquitinated protein). Forming proteins, signaling ligands, cell membrane permeabilizing peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (eg, CRISPR-Cas series, TALEN or zinc fingers), riboproteins, protein aptamers or chapelons. In some examples, increased expression in plants is about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60 relative to reference levels (eg, expression in untreated plants). %, 70%, 80%, 90%, 100% or more than 100% increased expression. In some examples, increased expression in plants is about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold, and about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold, about reference levels (expression in untreated plants). The expression is increased by 50 times, about 75 times, or about 100 times or more.

In some examples, the nucleic acid is an enzyme in an antisense RNA, disRNA, siRNA, shRNA, miRNA, aiRNA, PNA, morpholino, LNA, piRNA, ribozyme, DNAtime, aptamer (DNA, RNA), cyclRNA, gRNA or plant. DNA molecules (eg, antisense polynucleotides) that reduce the expression of metabolic enzymes, recombinase enzymes, helicase enzymes, integrase enzymes, RNAse enzymes, DNAse enzymes, polymerase enzymes, ubiquitinated proteins, superoxide control enzymes or energy producing enzymes). , Transcription factor, secretory protein, structural factor (actin, kinesin or tubulin), riboprotein, protein aptamer, chapelon, acceptor, signaling ligand or transport factor. In some examples, reduced expression in plants is about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, relative to reference levels (expression in untreated plants). Expression reduction of 70%, 80%, 90%, 100% or more than 100%. In some examples, reduced expression in plants is about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold relative to reference levels (eg, expression in untreated plants). , About 50 times, about 75 times, or about 100 times or more.

RNAi molecules contain sequences that are substantially complementary or completely complementary to all or fragments of the target gene. RNAi molecules can capture sequences at the boundary between introns and exons and prevent the maturation of newly generated RNA transcripts of a particular gene into mRNA for transcription. RNAi molecules complementary to a particular gene can hybridize with the mRNA of the target gene and block its translation. The antisense molecule can be DNA, RNA or a derivative or hybrid thereof. Examples of such derivative molecules include, but are not limited to, peptide nucleic acids (PNA) and phosphorothioate-based molecules such as deoxyribonucleic acid guanidine (DNG) or ribonucleic acid guanidine (RNG).

RNAi molecules can be provided as ready-to-use RNA synthesized in vitro or as an antisense gene transfected into cells that produce RNAi molecules during transcription. Hybridization with mRNA causes degradation of the hybrid molecule and / or inhibition of translation complex formation by RNAse H. Either way, the product of the gene of origin cannot be produced.

The length of the RNAi molecule that hybridizes to the transcript of interest is about 10 nucleotides, about 15-30 nucleotides or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, It can be 27, 28, 29, 30 nucleotides or longer. The degree of identity of the antisense sequence to the target transcript can be at least 75%, at least 80%, at least 85%, at least 90% or at least 95.

RNAi molecules can also contain overhangs, or typically unpaired protruding nucleotides, which are double helix structures normally formed by the core sequences of sense and antisense strand pairs as defined herein. Not directly involved in. The RNAi molecule can contain a 3'and / or 5'overhang of about 1-5 bases, independent of each of the sense and antisense strands. In some examples, both the sense and antisense strands contain 3'and 5'overhangs. In some examples, one or more 3'overhang nucleotides of one strand base pair with one or more 5'overhang nucleotides of the other strand. In another example, one or more 3'overhang nucleotides on one strand base do not pair with one or more 5'overhang nucleotides on the other strand. The sense and antisense strands of the RNAi molecule may or may not contain the same number of nucleotide bases. The antisense and sense strands have only the 5'ends with blunt ends, only the 3'ends with blunt ends, both the 5'and 3'ends with blunt ends, or the 5'and 3'. Neither of the ends can form a duplex that is not a blunt end. In another example, the one or more nucleotides in the overhang may contain thiophosphates, phosphorothioates, deoxynucleotide inverted (3'-3'linked) nucleotides, or modified ribonucleotides or deoxynucleotides.

Small interfering RNA (siRNA) molecules contain the same nucleotide sequence as about 15 to about 25 contiguous nucleotides of the target mRNA. In some examples, the siRNA sequence starts with dinucleotide AA and contains about 30-70% (about 30-60%, about 40-60% or about 45-55%) GC content, eg standard. As determined by the BLAST search, it does not contain a high identity percentage for any non-target nucleotide sequence in the genome into which it is introduced.

SiRNA and shRNA are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116: 281-297, 2004). In some examples, siRNAs can function as miRNAs and vice versa (Zeng et al., Mol. Cell 9: 1327-1333, 2002; Doench et al., Genes Dev. 17: 438. -442, 2003). External siRNAs downregulate mRNAs that have seed complementarity to siRNAs (Birmingham et al., Nat. Methods 3: 199-204, 2006). Multiple target sites within the 3'UTR provide stronger downregulation (Doench et al., Genes Dev. 17: 438-442, 2003).

Known valid siRNA sequences and allogeneic binding sites are also described in detail in the relevant literature. RNAi molecules are readily designed and made by techniques known in the art. In addition, there are computational tools that increase the chances of finding effective and specific sequence motifs (Pei et al., Nat. Methods 3 (9): 670-676, 2006; Reynolds et al., Nat. Biotechnol. 22 ( 3): 326-330, 2004; Khvorova et al., Nat. Struct. Biol. 10 (9): 708-712, 2003; Schwarz et al., Cell 115 (2): 199-208, 2003; Ui- Tei et al., Nucleic Acids Res. 32 (3): 936-948, 2004; Heale et al., Nucleic Acids Res. 33 (3): e30, 2005; 319 (1): 264-274, 2004; and Amarzguioui et al., Biochem. Biophys. Res. Nucleic Acid. 316 (4): 1050-1058, 2004).

The RNAi molecule regulates the expression of RNA encoded by the gene. In some examples, RNAi molecules can be designed to target a class of genes with sufficient sequence homology, as multiple genes can share some degree of sequence homology with each other. In some examples, RNAi molecules may contain sequences that are shared between different gene targets or that have complementarity to a sequence that is unique for a particular gene target. In some examples, the RNAi molecule targets conserved regions of RNA sequences that are homologous to several genes, thereby allowing several genes within the gene family (eg, different gene isoforms, splice variants). , Mutant genes, etc.) can be designed to be targeted. In some examples, RNAi molecules can be designed to target a unique sequence for a particular RNA sequence of a single gene.

Inhibiting RNA molecules include, for example, modified nucleotides such as 2'-fluoro, 2'-o-methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, 2 '-Can be modified to contain deoxyuridine. Without being bound by theory, it is believed that such modifications may increase nuclease resistance and / or serum stability or reduce immunogenicity.

In some examples, the RNAi molecule is linked to the delivery polymer via a physiologically unstable bond or linker. Physiologically unstable linkers are selected to undergo chemical transformations (eg, cleavage) when they are present under certain physiological conditions (eg, disulfide bonds are cleaved in the reducing environment of the cytoplasm). Will be). The release of molecules from the polymer by breaking physiologically unstable bonds promotes the interaction of the molecules with the appropriate cell components for activity.

RNAi molecule-polymer conjugates can be formed by covalently bonding the molecule to the polymer. The polymer is polymerized or modified so that it contains the reactive group A. The RNAi molecule is also polymerized or modified so that it contains a reactive group B. Reactive groups A and B are selected so that they can be linked via reversible covalent bonds using methods known in the art.

The conjugate of the RNAi molecule to the polymer can be carried out in the presence of excess polymer. Since RNAi molecules and polymers can be countercharged during conjugation, the presence of excess polymer can reduce or eliminate conjugate aggregation. Alternatively, excess carrier polymers such as polycations can be used. The excess polymer can be removed from the conjugated polymer prior to administration of the conjugate. Alternatively, the excess polymer can be co-administered with the conjugate.

The production and use of inhibitors based on non-coding RNAs such as ribozymes, RNAse Ps, siRNAs and miRNAs have also been described, for example, in Side, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). As described in Humana Press (2010), it is known in the art.

(G) Gene Editing The plant modification compositions described herein may contain elements of the gene editing system. For example, the agent can introduce a modification (eg, insertion, deletion (eg, knockout), translocation, inversion, single point mutation or other mutation) into one gene in a plant. Exemplary gene editing systems include zinc finger nucleases (ZFNs), transcriptional activator-like effector-based nucleases (TALENs), and clustered, regularly spaced short-chain palindromic sequence repeat (CRISPR) systems. Methods based on ZFNs, TALENs and CRISPR are described, for example, in Gaj et al. , Trends Biotechnol. 31 (7): 397-405, 2013.

In a typical CRISPR / Cas system, a sequence-specific, non-coding guide RNA that targets a single- or double-stranded DNA sequence allows the endonuclease to be a target nucleotide sequence (eg, within the genome to be sequence-edited). Part). Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One Class II CRISPR system includes Type II Cas endonucleases such as Cas9, CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA). The crRNA contains a guide RNA, i.e., typically an approximately 20 nucleotide RNA sequence corresponding to the target DNA sequence. The crRNA also includes a region that binds to tracrRNA, forming a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid. RNA acts as a guide to direct the Cas protein and suppress specific DNA / RNA sequences, depending on the spacer sequence. For example, Horvas et al. , Science 327: 167-170, 2010; Makarova et al. , Biology Direct 1: 7, 2006; Pennisi, Science 341: 833-836, 2013. The target DNA sequence generally must be flanking a protospacer flanking motif (PAM) specific for a given Cas endonuclease; the PAM sequence appears throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; 5'-NGG (SEQ ID NO: 1) (Streptococcus pyogenes), 5', as an example of PAM sequences. -NNAGAA (SEQ ID NO: 2) (Streptococcus thermophilus CRISPR1), 5'-NGGNG (SEQ ID NO: 3) (Streptococcus thermophilus (Streptococcus thermophilus) CRISPR3) ) (Neisseria meningitidis). Some endonucleases, such as Cas9 endonucleases, bind to the G-rich P (5'side) AM site, such as 5'-NGG (SEQ ID NO: 1), and target DNA at a position 3 nucleotides upstream from the PAM site. Perform blunt-ended cleavage. Another Class II CRISPR system contains a V-type endonuclease Cpf1, which is smaller than Cas9; for example, AsCpf1 (derived from the genus Acidaminococcus sp.) And LbCpf1 (Lachnospiraceae sp. ) Origin). Cpf1-binding CRISPR arrays are processed into mature crRNA without the requirement of tracrRNA; in other words, the Cpf1 system requires only Cpf1 nuclease and crRNA to cleave the target DNA sequence. The Cpf1 endonuclease is associated with a T-rich PAM site, such as 5'-TTN (SEQ ID NO: 5). Cpf1 also recognizes the 5'-CTA (SEQ ID NO: 6) PAM motif. Cpf1 cleaves DNA by introducing an offset or staggered double-strand break containing a 4'-or 5-nucleotide 5'overhang, eg, 18 nucleotides downstream from (3' side of) the PAM site of the coding strand. And the target DNA is cleaved with a 5-nucleotide offset or staggered cleavage located 23 nucleotides downstream from the PAM site of the complementary strand, and the 5-nucleotide overhang resulting from such offset cleavage is blunt-ended by DNA insertion by homologous recombination. Allows for more accurate genome editing than by insertion with truncated DNA. For example, Zetsche et al. , Cell 163: 759-771, 2015.

For gene editing purposes, CRISPR arrays can be designed to contain one or more guide RNA sequences corresponding to the desired target DNA sequence; eg, Cong et al. , Science 339: 819-823, 2013; Ran et al. , Nature Protocols 8: 2281-2308, 2013. At least about 16 or 17 nucleotides of the gRNA sequence are required to cause DNA cleavage by Cas9; in the case of Cpf1, at least about 16 nucleotides of the gRNA sequence are required to perform detectable DNA cleavage. .. In fact, guide RNA sequences are generally designed to have a length of 17-24 nucleotides (eg, 19, 20 or 21 nucleotides) and complementarity with the targeted gene or nucleic acid sequence. Custom gRNA production devices and algorithms are commercially available for use in the design of effective guide RNAs. Gene editing mimics a chimeric single-guide RNA (sgRNA), a naturally occurring crRNA-tracrRNA complex, and induces a nuclease with tracrRNA (for binding to a nuclease) and at least one crRNA (the sequence targeted for editing). It has also been achieved using an engineered (synthetic) single RNA molecule containing both (to) and. Chemically modified sgRNAs have also been demonstrated to be effective in genome editing; for example, Handel et al. , Nature Biotechnology. See 985-991, 2015. In some examples, heterologous plant modifiers are ribs containing one or more RNA molecules, such as gRNA or sgRNA, and one or more RNA binding proteins, such as endonucleases, such as Casendonucleases (eg, Cas9 endonucleases). Contains nuclear protein complex (RNA).

Wild Cas9 produces double-strand breaks (DSBs) at specific DNA sequences targeted by gRNAs, but several CRISPR endonucleases with modified functionality are available, such as Cas9. The nickase version produces only single-strand breaks; non-catalytic Cas9 (dCas9) does not cut the target DNA, but interferes with transcription by steric damage. dCas9 can also be further fused with an effector to suppress (CRISPRi) or activate (CRISPRa) the expression of the target gene. For example, Cas9 can be fused with a transcriptional repressor (eg, KRAB domain) or a transcriptional activator (eg, dCas9-VP64 fusion). Non-catalytic Cas9 (dCas9) (dCas9-Fokl) fused with Fokl nuclease can be used to generate DSBs with target sequences homologous to the two gRNAs. See, for example, the numerous CRISPR / Cas9 plasmids disclosed in the Addgene repository (Addgene, 75 Sidney St., Suite 550A, Cambridge, MA 02139; addgene.org/crispr /) and available from which. The double nickase Cas9, each introducing two separate double-strand breaks directed by a separate guide RNA, is described in Ran et al. , Cell 154: 1380-1389, 2013, described as achieving more accurate genome editing.

CRISPR technology for editing eukaryotic genes includes U.S. Patent Application Publication Nos. 2016/013808 A1 and 2015/0344912 A1 and U.S. Pat. Nos. 8,697,359. 8,771,945, 8,945,839, 8,999,641 and 8,993,233, 8,895,308. No. 8,865,406, No. 8,889,418, No. 8,871,445, No. 8,889,356, No. It is disclosed in the specification of No. 8,923,814, the specification of No. 8,795,965 and the specification of No. 8,906,616. The Cpf1 endonuclease and the corresponding guide RNA and PAM site are disclosed in US Patent Application Publication No. 2016-0208243 A1.

In some examples, the desired genomic modification involves homologous recombination, in which case the homologous set after RNA-induced nucleases and guide RNAs generate one or more double-stranded DNA breaks in the target nucleotide sequence. Repair of cleavage using a conversion mechanism (homologous recombination repair) occurs. In these examples, donor templates encoding the desired nucleotide sequence to be inserted or knocked in at double-strand breaks are provided to the cell or subject; examples of suitable templates are single-stranded DNA templates and double-stranded DNA. Examples include templates (eg, linked to the polypeptides described herein). Generally, donor templates encoding nucleotide changes over regions less than about 50 nucleotides are provided in the form of single-stranded DNA; larger donor templates (eg, greater than 100 nucleotides) are often double-stranded DNA plasmids. Provided as. In some examples, the donor template is sufficient to achieve the desired homologous recombination repair, but does not persist in the cell or subject after a given period (eg, after one or more cell division cycles). In quantity, it is fed to the cell or subject. In some examples, the donor template has a core nucleotide sequence that differs from the target nucleotide sequence by at least one, at least five, at least 10, at least 20, at least 30, at least 40, at least 50 or more nucleotides (eg, homology). It has an endogenous genomic region). This core sequence is flanked by homology arms or regions of high sequence identity with the target nucleotide sequence; in some examples, the regions of high sequence identity are at least 10, at least 50, at least on either side of the core sequence. Includes 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750 or at least 1000 nucleotides. In some examples where the donor template is in the form of single-stranded DNA, the core sequences are at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80 on either side of the core sequence. Alternatively, it is flanked by a homology arm containing at least 100 nucleotides. In some examples where the donor template is in the form of double-stranded DNA, the core sequence is a homology arm comprising at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 nucleotides on either side of the core sequence. Franked by. In one example, double nickase Cas9 was used to introduce two separate double-strand breaks into a cell or target nucleotide sequence of interest (see Ran et al., Cell 154: 1380-1389, 2013). , Donor template delivery is performed.

In some examples, the composition contains gRNAs and target nucleases such as Cas9, such as wild-type Cas9, nickase Cas9 (eg Cas9D10A), inactive Cas9 (dCas9), eSpCas9, Cpf1, C2C1 or C2C3 or such nucleases. Contains the encoding nucleic acid. The choice of nuclease and gRNA is determined by whether the target mutation is a nucleotide deletion, substitution or addition, eg, a nucleotide deletion, substitution or addition to the target sequence. By fusion of all or part of (one or more) effector domains (eg, biologically active moieties) with tethered non-catalytic endonucleases, such as inactive Cas9 (dCas9, eg D10A; H840A). , Creating a chimeric protein and ligating it with a polypeptide to direct the composition to a particular DNA site by one or more RNA sequences (sgRNAs) and the activity and / or of one or more target nucleic acid sequences. Expression can be regulated. In some examples, gRNAs and targeting nucleases are provided as ribonuclear protein complexes (RNPs).

In multiple examples, the agent comprises a guide RNA (gRNA) used in the CRISPR system for gene editing purposes. In some examples, the agent comprises a zinc finger nuclease (ZFN) or an mRNA encoding a ZFN that targets (eg, cleaves) a nucleic acid sequence (eg, DNA sequence) of one gene in a plant. In some examples, the agent comprises TALEN or an mRNA encoding TALEN that targets (eg, cleaves) a nucleic acid sequence (eg, DNA sequence) of one gene in a plant.

For example, gRNA can be used in the CRISPR system to manipulate genetic modification in plants. In another example, ZFNs and / or TALENs can be used to engineer genetic modification in plants. Exemplary modifications include insertions, deletions (eg, knockouts), translocations, inversions, single point mutations or other mutations. Modifications can be introduced into the gene of the cell, eg, in vitro, ex vivo or in vivo. In some examples, the modification increases the level and / or activity of the gene in the plant. In another example, the modification reduces the level and / or activity of the gene in the plant (eg, knocks down or knocks out). In yet another example, the modification corrects a genetic defect (eg, a mutation that causes the defect) in a plant.

In some examples, the CRISPR system is used to edit target genes in plants (eg, add or delete base pairs). In another example, the CRISPR system is used to introduce an immature stop codon, eg, thereby reducing expression of the target gene. In yet another example, the CRISPR system is used to turn off the target gene in a reversible manner, eg, RNA interference. In some examples, a CRISPR system is used to direct Cas to the promoter of the gene, thereby sterically blocking RNA polymerase.

In some examples, the CRISPR system is described, for example, in US Patent Application Publication No. 20140068977, Kong, Science 339: 819-823, 2013; Tsai, Nature Biotechnology. 32: 6 569-576, 2014; U.S. Pat. No. 8,871,445; U.S. Pat. No. 8,865,406; No. 8,795,965; No. 8,771,945. No.; and the techniques described in No. 8,697,359 can be used to generate genes for editing in plants.

In some examples, CRISPR interference (CRISPRi) technology can be used for transcriptional repression of specific genes in plants. In the case of CRISPRi, an engineered Cas9 protein (eg, a nuclease-null dCas9 or dCas9 fusion protein, such as a dCas9-KRAB or dCas9-SID4X fusion) can be paired with a sequence-specific guide RNA (sgRNA). The Cas9-gRNA complex can block RNA polymerase, thereby interfering with transcription elongation. This complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method has a minimal off-target effect uniquely and is multiplexable, for example, it can suppress two or more genes at the same time (eg, using multiple gRNAs). The CRISPRi method also allows for reversible gene suppression.

In some examples, CRISPR-mediated gene activation (CRISPRa) can be used, for example, for transcriptional activation of plant genes. In CRISPRa technology, the dCas9 fusion protein recruits transcriptional activators. For example, dCas9 is fused with a polypeptide such as a VP64 or p65 activation domain (p65D) (eg, an activation domain) and used in conjunction with an sgRNA (eg, a single sgRNA or multiple sgRNAs) of a plant. One or more genes can be activated. By using multiple sgRNAs, multiple activators can be mobilized, which can increase activation efficiency. Various activation domains and one or more activation domains can be used. In addition to manipulating dCas9 to mobilize activators, sgRNAs can also be manipulated to mobilize activators. For example, by incorporating an RNA aptamer into an sgRNA, a protein such as VP64 (eg, an activation domain) can be introduced. In some examples, synergistic activation mediator (SAM) systems can be used for transcriptional activation. In SAM, the MS2 aptamer is added to the sgRNA. MS2 recruits MS2 coated protein (MCP) fused with p65AD and heat shock factor 1 (HSF1).

For more details on CRISPRi and CRISPRa techniques, see Dominguez et al. , Nat. Rev. Mol. Cell Biol. 17: 5-15, 2016 (incorporated herein by reference). In addition, Dominguez et al. As described in ,, dCas9-mediated epigenetic modification and co-activation and suppression by the CRISPR system can be used to regulate genes in plants.

V. Kit The present invention also provides a kit for plant modification, which kit comprises a container with the plant modification composition described herein. The kit may further include teaching material for applying or delivering (eg, to a plant) a plant modification composition for modifying a plant according to the method of the invention. Those skilled in the art will appreciate that the description in applying the plant modification composition by the method of the present invention can be any form of description. Such instructions include, but are not limited to, documentary materials (eg, labels, booklets, pamphlets), oral materials (eg, cassette tapes or CDs) or video materials (eg, videotapes or DVDs).

The following is an example of the method of the present invention. It is understood that various other embodiments may be implemented in view of the outline provided above.

Example 1: Isolation of Plant Messenger Packs from Plants This example contains a variety of plant origins, including leaf apoplasts, seed apoplasts, roots, fruits, vegetables, pollen, phloem sap, xylem sap and plant cell media. The isolation of crude plant messenger packs (PMPs) from.

Experimental design:
a) PMP isolation from Arabidopsis apoplast leaves of Arabidopsis thaliana Arabidopsis (Arabidopsis thaliana Cl-0) seeds are surface sterilized with 50% bleaching agent and then surface sterilized with 0.8% agar. .53 Plate culture on Murashige and Skoog medium. After vernalizing these seeds at 4 ° C for 2 days, they are transferred to short-day conditions (light period 9h, 22 ° C, 150 μEm- ^2). After 1 week, transfer the seedlings to Pro-Mix PGX. The plants are grown for 4-6 weeks and then harvested.

Plant Physiol. 173 (1): PMP is isolated from the apoplast lavage fluid of Arabidopsis rosettes 4-6 weeks old as described in 728-741, 2017. Briefly, whole rosettes are harvested from the roots and vacuum infiltrated with vesicle isolation buffer (20 mM MES, 2 mM CaCl2 and 0.1 M NaCl, pH 6). Collect the EV-containing apoplast extracellular fluid by carefully blotting the infiltrated plant to remove excess liquid, introducing it into a 30 mL syringe, and then centrifuging at 2 ° C. for 20 minutes in a 50 mL conical tube. do. Next, the extracellular fluid of apoplast is filtered through a 0.85 μm filter to remove large particles, and then the PMP is purified as described in Example 2.

b) PMP isolation from sunflower seed apoplasts Intact sunflower seeds (sunflower (H. annuus L.) were allowed to absorb water for 2 hours, peeled and peeled, and then Regente et al, FEBS Letters. Extract the apoplast extracellular fluid by a modified vacuum infiltration-centrifugation procedure modified from 583: 3363-3366, 2009. Briefly, seeds are vesicle isolated buffer (20 mM MES, 2 mM CaCl2 and 0.1 M NaCl, After infiltration into pH 6), the seeds are subjected to three 10-second vacuum pulses and separated by a pressure of 45 kPa at 30-second intervals. The infiltrated seeds are collected, dried on filter paper, and placed in a frit glass filter. Then, centrifuge at 400 g at 4 ° C. for 20 minutes. After collecting the apoplast extracellular solution and filtering with a 0.85 μm filter to remove large particles, the PMP is purified as described in Example 2. do.

c) PMP isolation from root ginger Fresh ginger (Zingiber office) rhizome roots are purchased from a local supplier and washed 3 times with PBS. A total of 200 grams of washed roots were crushed with a mixer (Osterizer 12-speed blender) at a maximum speed of 10 minutes (1 minute rest every 1 minute of blending), and then Zhuang et al. , J Extracellular Vesicles. 4 (1): PMP is isolated as described in 28713, 2015. Briefly, large particles are removed from the PMP-containing supernatant by sequentially centrifuging the ginger juice at 1,000 g for 10 minutes, 3,000 g for 20 minutes and 10,000 g for 40 minutes. Purify the PMP as described in Example 2.

d) PMP isolation from grapefruit juice Fresh grapefruit (Grapefruit (Citrus x paradisi)) was purchased from a local supplier, its skin was removed, and Wang et al. , Molecular Therapy. 22 (3): Fruits are squeezed by hand (with minor modifications) as described in 522-534, 2014, or blended for 10 minutes at maximum speed with a mixer (Osterizer 12-speed blender). Collect fruit juice by crushing (pause for 1 minute every 1 minute). Briefly, large particles are removed from the PMP-containing supernatant by sequentially centrifuging the juice / juice pulp at 1,000 g for 10 minutes, 3,000 g for 20 minutes and 10,000 g for 40 minutes. Purify the PMP as described in Example 2.

e) Isolation of PMP from broccoli head Broccoli (Broccoli (Brassica oleracea var. Italica)) PMP has been previously described (Deng et al., Molecular Therapy, 25 (7): 1641-1654, 2017). Isolate. Briefly, fresh broccoli is purchased from a local supplier, washed 3 times with PBS and then crushed with a mixer (Osterizer 12-speed blender) at maximum speed for 10 minutes (1 minute rest every 1 minute of blending). do. Next, large particles are removed from the PMP-containing supernatant by sequentially centrifuging the broccoli juice at 1,000 g for 10 minutes, 3,000 g for 20 minutes and 10,000 g for 40 minutes. Purify the PMP as described in Example 2.

f) Isolation of PMP from olive pollen Olive (Olea europaea) pollen EV is described in Prado et al. , Molecular Plant. 7 (3): Isolate as previously described in 573-577, 2014. Briefly, olive pollen (0.1 g) was hydrated in a humidified chamber at room temperature for 30 minutes, then 20 ml of germination medium: 10% sucrose, 0.03% Ca (NO ₃ ) ₂ , 0.01%. Transfer to a Petri dish (15 cm in diameter) containing KNO ₃ , 0.02% ו ₄ and 0.03% H ₃ BO _3. Pollen is germinated in a dark place at 30 ° C. for 16 hours. Pollen grains are considered to germinate only when the tube is longer than the diameter of the pollen grains. Medium containing PMP is collected and pollen debris removed by two consecutive filtrations with a 0.85 um filter by centrifugation. Purify the PMP as described in Example 2.

g) PMP isolation from Arabidopsis tubules Arabidopsis (Arabidopsis thaliana Col-0) seeds are surface sterilized with 50% bleach and then contain 0.8% agar. 53 Plate culture on Murashige and Skoog medium. After vernalizing these seeds at 4 ° C for 2 days, they are transferred to short-day conditions (light period 9h, 22 ° C, 150 μEm- ^2). After 1 week, transfer the seedlings to Pro-Mix PGX. The plants are grown for 4-6 weeks and then harvested.

Tetyuki et al. , By JOVE. As described in 80, 2013, phloem fluid from Arabidopsis rosette leaves aged 4 to 6 weeks is collected. Briefly, leaves are cut from the base of the petiole, stacked and then placed in the dark for 1 hour in a reaction tube containing 20 mM K2-EDTA to prevent wound sealing. The leaves are gently removed from the container, thoroughly washed with distilled water, all EDTA is removed, placed in a clean tube, and then the tube solution is collected in the dark over 5-8 hours. The leaves are discarded and the phloem solution is filtered through a 0.85 μm filter to remove large particles before purifying the PMP as described in Example 2.

h) PMP isolation from tomato plant Kibe sap Tomato (Solanum lycopercium) seeds are planted in a single pot containing organic soil such as Sunshine Mix (Sun Greenhouse, Agawam, MA) and 22 ° C to 28 ° C. Keep in a greenhouse at ℃. Approximately 2 weeks after germination, that is, at the two-leaf stage (two true-leaf stage), seedlings are placed in pots (10 cm in diameter and 17 cm in depth) filled with sterile sandy soil containing 90% sand and a 10% organic matter mix. Port individually. Keep the plants in a greenhouse at 22 ° C to 28 ° C for 4 weeks.

Coal et Al. , Plant Physiology. As described in 155 (2): 974-987,2011, xylem sap from 4-week-old tomato plants is collected. Briefly, cut above the hypocotyl of the tomato plant and attach a plastic ring around the stem. The xylem sap that accumulates over 90 minutes after cutting is collected. The xylem sap is filtered through a 0.85 μm filter to remove large particles before purifying the PMP as described in Example 2.

i) Isolation of PMP from tobacco BY-2 cell medium 30 g / L sucrose, 2.0 mg / L potassium dihydrogen phosphate, 0.1 g / L myo-inositol, 0.2 mg / L 2,4 -Shaker at 180 rpm in MS (Murashige and Skoog, 1962) BY-2 medium (pH 5.8) containing MS salt (Duchefa, Harlem, Netherlands, # M0221) supplemented with dichlorophenoxyacetic acid and 1 mg / L thiamine HCl. Tobacco BY-2 (Tobacco (Nicotiana tabacum L cv.) Bright Yellow 2) cells are cultured in a dark place at 26 ° C. BY-2 cells are secondarily cultured weekly by transferring 5% (v / v) of the 7-day-old cell culture to 100 mL of fresh liquid medium. After 72-96 hours, the BY-2 medium is collected and the cells are removed by centrifugation at 4 ° C., 300 g for 10 minutes. The supernatant containing PMP is collected and filtered through a 0.85 um filter to remove debris. Purify the PMP as described in Example 2.

Example 2: Generation of Purified Plant Messenger Pack (PMP) This example is of size exclusion chromatography, in combination with a density gradient (iodixanol or sucrose), with ultrafiltration and precipitation or size exclusion chromatography of aggregates. The production of purified PMP from the crude PMP fraction according to Example 1 using removal will be described.

Experimental design:
a) Generation of purified grapefruit PMP using ultrafiltration in combination with size exclusion chromatography A crude grapefruit PMP fraction from Example 1a using a 100-kDA molecular weight cutoff (MWCO) American spin filter (Merck Millipore). To concentrate. The concentrated crude PMP solution is then loaded onto a PURE-EV size exclusion chromatography column (HansaBioMed Life Sciences Ltd) and isolated according to the manufacturer's instructions. The purified PMP-containing fraction is pooled after elution. Optionally, PMP can also be further concentrated using a 100-kDA MWCO Amion spin filter or by tangential flow filtration (TFF). Purified PMP is analyzed as described in Example 3.

b) Generation of purified Arabidopsis apoplast PMP using iodixanol gradient As described in Example 1a, crude Arabidopsis leaf apoplast PMP was isolated from Rutter and Innes, Plant Physiol. 173 (1): Purified PMP is produced as described in 728-741, 2017. Diluting an aqueous 60% OptiPrep stock solution with vesicle isolation buffer (VIB; 20 mM MES, 2 mM CaCl2 and 0.1 M NaCl, pH 6) to prepare a discontinuous Iodixanol gradient (OptiPrep; Sigma-Aldrich). To prepare solutions of 40% (v / v), 20% (v / v) and 5% (v / v) iodixanol. The gradient is formed by laminating 3 ml of 40% solution, 3 mL of 20% solution, 3 mL of 10% solution and 2 mL of 5% solution. The crude apoplast PMP solution from Example 1a is centrifuged at 4 ° C. and 40,000 g for 60 minutes. The pellet is resuspended in 0.5 ml VIB and then layered on top of the gradient. Centrifuge for 17 hours at 4 ° C. and 100,000 g. After discarding the first 4.5 ml at the top of the gradient, collect 3 volumes of 0.7 ml containing apoplast PMP, make 3.5 mL with VIB and centrifuge at 4 ° C. and 100,000 g for 60 minutes. do. The pellet is washed with 3.5 ml VIB and then re-pelletized using the same centrifugation conditions. Purified PMP pellets are combined for the next analysis as described in Example 3.

c) Generation of purified grapefruit PMP using sucrose gradient A crude grapefruit juice PMP was isolated as described in Example 1d, centrifuged at 150,000 g for 90 minutes, and then described (Mu et Al.,. PMP-containing pellets are resuspended in 1 ml of PBS as in Molecular Nutrition & Food Research.58 (7): 1561-1573, 2014). The resuspended pellet is transferred to a sucrose step gradient (8% / 15% / 30% / 45% / 60%) and centrifuged at 150,000 g for 120 minutes to produce purified PMP. Purified grapefruit PMP is collected from the 30% / 45% interface and later analyzed as described in Example 3.

d) Removal of Aggregates from Grapefruit PMP Additional purification steps can be added to remove protein aggregates from the grapefruit PMPs produced as described in Example 1d or the purified PMPs from Examples 2a-c. .. The resulting PMP solution is passed through various pH to precipitate protein aggregates in the solution. The pH is adjusted to 3, 5, 7, 9 or 11 by adding sodium hydroxide or hydrochloric acid. The pH is measured using a calibrated pH probe. When the solution reaches the specified pH, it is filtered to remove the particles. Alternatively, the isolated PMP solution can be condensed by the addition of a charged polymer such as Polymin-P or Plastol 2640. Briefly, 2-5 g of Polymin-P or Plastol 2640 per liter is added to the solution and mixed with an impeller. The solution is then filtered to remove the particles. Instead, the aggregates are solubilized by increasing the salt concentration. NaCl is added to the PMP solution until it reaches 1 mol / L. The solution is then filtered to purify the PMP. Instead, the agglomerates are solubilized by increasing the temperature. The isolated PMP mixture is heated for 5 minutes with mixing until a homogeneous temperature of 50 ° C. is reached. The PMP mixture is then filtered to isolate the PMP. Instead, soluble contaminants from the PMP solution are separated by a size exclusion chromatography column according to standard methods, in which case the PMP elutes to the first fraction, whereas the protein and ribonucleoprotein and Some lipoproteins are delayed elution. The efficiency of protein aggregate removal is determined by measuring and comparing protein concentrations before and after protein aggregate removal by BCA / Bladeford protein quantification. The generated PMP is analyzed as described in Example 3.

Example 3: Derivativeization of Plant Messenger Pack This example describes the characterization of PMP produced as described in Example 1 or Example 2.

Experimental design:
a) Determination of PMP concentration PMP particle concentration is determined by Nanoparticle Tracking Analysis (NTA) using Malvern NanoSight or Tunable Resistive Pulse Sen (Tunable Resistive Pulse) by iZon qNano. Follow the instructions in. The protein concentration of purified PMP is determined by the use of the DC protein assay (Bio-Rad). The lipid concentration of the purified PMP was determined by Plant Physiol. 173 (1): Determined using a fluorescent lipophilic dye such as DiOC6 (ICN Biomedicals) as described in 728-741, 2017. Briefly, 100 ml of 10 mM DiOC6 (ICN Biomedicals) diluted with MES buffer (20 mM MES, pH 6) the purified PMP pellets from Example 2 and 1% plant protease inhibitor cocktail (Sigma-Aldrich) and 2 mM 2,29. -Resuspend in dipyridyl disulfide. Resuspend PMP is incubated at 37 ° C. for 10 minutes, washed with 3 mL MES buffer, repelletized (400,000 g at 4 ° C., 60 minutes) and resuspended in fresh MES buffer. The DiOC6 fluorescence intensity is measured with 485 nm excitation and 535 nm emission.

b) Biophysical and molecular property determination of PMP Wu et Al. , Analyst. 140 (2): PMP is characterized by electron and cryo-electron microscopy in a JEOL 1010 transmission electron microscope according to the protocol of 386-406, 2015. Using Malvern Zetasizer or iZon qNano, the size of PMP and zeta potential are also measured according to the manufacturer's instructions. Lipids were isolated from PMP using chloroform extraction and Xiao et al. Plant Cell. 22 (10): As described in 3193-3205, 2010, the characteristics are determined by LC-MS / MS. Glycosyl inositol phosphoryl ceramide (GIPC) lipids were extracted from Cass et al. , Plant Physiology. After purification as described in 170: 367-384, 2016, analysis is performed by LC-MS / MS as described above. Using the Quant-It kit from Thermo Fisher, the total RNA, DNA and protein are characterized according to the instructions. Rutter and Innes, Plant Physiol. 173 (1): PMP proteins are characterized by LC-MS / MS according to the protocol described in 728-741, 2017. RNA and DNA were extracted using Trizol and prepared into a library containing TruSeq Total RNA using Illumina's Ribo-Zero Plant kit and Nextera Mate Pair Library Prep Kit, and then according to the manufacturer's instructions. , Illumina MiSeq.

Example 4: Determining the Stability of the Plant Messenger Pack This example illustrates the measurement of the stability of PMP under various storage and physiological conditions.

Experimental design:
The PMPs generated as described in Examples 1 and 2 are subject to various conditions. Suspension PMP in water, 5% sucrose or PBS and leave at −20 ° C., 4 ° C., 20 ° C. and 37 ° C. for 1, 7, 30 and 180 days. Also, the PMP is suspended in water, dried using a rotary evaporator device, and left at 4 ° C, 20 ° C and 37 ° C for 1, 7 and 30 and 180 days. In addition, PMP was suspended in water or a 5% sucrose solution, quickly frozen in liquid nitrogen, and then freeze-dried. After 1, 7, 30 and 180 days, the dried and lyophilized PMPs are resuspended in water. The three prior experiments, including conditions at temperatures above 0 ° C., are also exposed to artificial solar simulators to determine satisfactory safety in simulated outdoor UV conditions. In addition, 1 unit of trypsin added or not added to pH 1, 3, 5, 7 and 9 buffer solutions or other artificial gastric juices at 37 ° C, 40 ° C, 45 ° C, 50 ° C and 55 ° C. Expose PMP to temperature for 1, 6 and 24 hours.

After each of these treatments, the PMP is returned to 20 ° C., neutralized to pH 7.4, and then characterized using some or all of the methods described in Example 3.

Example 5. Loading PMPs by Cargo This example describes a method of loading PMPs using small molecules, proteins and nucleic acids.

a) Loading small molecules into the PMP PMPs are generated as described in Examples 1 and 2. To load the small molecule into the PMP, the PMP is placed in a PBS solution containing the small molecule, either in solid form or in solubilized form. This solution was prepared by Sun, Mol. The. , 2010, leave at 22 ° C. for 1 hour. Instead, Wang et al, Nature Comm. , 2013, sonicate the solution to induce poration and diffusion into exosomes. Instead, PMP is described in Wahlgren et al, Nucl. Acids. Res. Electropolate according to the protocol from 2012.

Alternatively, PMP lipids are isolated and vortexed by adding 3.75 ml of 2: 1 (v / v) MeOH: CHCl3 to 1 ml of PMP in PBS. CHCl3 (1.25 ml) and ddH2O (1.25 ml) are added sequentially and vortexed. The mixture is then centrifuged in a glass tube at 22 ° C. at 2,000 rpm for 10 minutes to separate the mixture into two phases (aqueous phase and organic phase). Organic layer samples containing PMP lipids are dried by heating under nitrogen (2 psi). To generate a small molecule load PMP, the isolated PMP lipids were mixed with a small molecule solution and Haney et al, J Control. Rel. , 2015, pass the solution through the lipid extruder.

Prior to use, the loaded PMP is purified using the method described in Example 2 to remove unbound small molecules. Load PMPs are characterized as described in Example 3 and their stability is tested as described in Example 4.

b) Loading the protein or peptide into the PMP PMPs are produced as described in Examples 1 and 2. To load the protein or peptide into the PMP, the PMP is placed in solution with the protein or peptide in PBS. If the protein or peptide is insoluble, adjust the pH until it is soluble. If the protein or peptide is still insoluble, use the insoluble protein or peptide. Next, Wang et al, Nature Comm. , 2013, sonicate the solution to induce poration and diffusion in the PMP. Instead, Wahlgren et al, Nucl. Acids. Res. Electroporate PMP according to the protocol from 2012.

Alternatively, PMP lipids are isolated by adding 3.75 ml of 2: 1 (v / v) MeOH: CHCl3 to 1 ml of PMP in PBS. CHCl3 (1.25 ml) and ddH2O (1.25 ml) are added sequentially and vortexed. The mixture is then centrifuged in a glass tube at 22 ° C. at 2,000 rpm for 10 minutes to separate the mixture into two phases (aqueous phase and organic phase). Organic layer samples containing PMP lipids are dried by heating under nitrogen (2 psi). To generate a small molecule load PMP, the isolated PMP lipids were mixed with a small molecule solution and Haney et al, J Control. Rel. , 2015, pass the solution through the lipid extruder.

Prior to use, the loaded PMP is purified using the method described in Example 2 to remove unbound peptides and proteins. Load PMPs are characterized as described in Example 3 and their stability is tested as described in Example 4. To measure protein or peptide loading, use the Pierce Quantitative Colorimetric Peptide Assay for small samples of loaded and unloaded PMP.

c) Loading Nucleic Acids into PMPs PMPs are produced as described in Examples 1 and 2. To load the nucleic acid into the PMP, the PMP is placed in solution with the nucleic acid in PBS. Next, Wang et al, Nature Comm. , 2013, sonicate the solution to induce poration and diffusion in the PMP. Instead, Wahlgren et al, Nucl. Acids. Res. Electroporate PMP according to the protocol from 2012.

Alternatively, PMP lipids are isolated by adding 3.75 ml of 2: 1 (v / v) MeOH: CHCl3 to 1 ml of PMP in PBS. CHCl3 (1.25 ml) and ddH2O (1.25 ml) are added sequentially and vortexed. The mixture is then centrifuged in a glass tube at 22 ° C. at 2,000 rpm for 10 minutes to separate the mixture into two phases (aqueous phase and organic phase). Organic layer samples containing PMP lipids are dried by heating under nitrogen (2 psi). To generate a small molecule load PMP, the isolated PMP lipids were mixed with a small molecule solution and Haney et al, J Control. Rel. , 2015, pass the solution through the lipid extruder.

Prior to use, PMP is purified using the method described in Example 2 to remove unbound nucleic acid. Load PMPs are characterized as described in Example 3 and their stability is tested as described in Example 4. Nucleic acid loaded into the PMP is quantified using the Thermo Fisher Quant-It assay according to the manufacturer's instructions, or whether the nucleic acid is fluorescently labeled using a plate reader.

Example 6: Modification of plants by PMP-mediated plasmid delivery in plants This example describes loading and functional delivery of plasmid-loaded PMPs in plants to affect the fitness of plants. This example also further demonstrates that the plasmid loaded PMP is stable and retains its activity over a variety of processing and environmental conditions.

In this example, cotton is used as a model plant, grapefruit PMP is used as a model PMP, and pCambia1303 GUSA: mGFP5 reporter expression vector is used as a model plasmid.

Treatment plan:
Grapefruit PMP solution is formulated with equal doses of 0, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml plasmids in sterile water.

Experimental protocol:
Loading the GUSA: mGFP5 reporter plasmid into the grapefruit PMP The PMP is produced from grapefruit as described in Examples 1 and 2. To demonstrate functional plasmid delivery by PMP, the pCambia1303 reporter plasmid (12,361bp; Marker Gene) expressing the fused gusA: mgfp5 reporter gene driven by a double-enhanced version of the CaMV35S promoter on grapefruit PMP and terminated by the CaMV35S polyA signal. Loaded by (purchased from Technologies). Upon transcription, both GUSA and mGFP5 are transcribed and can be detected. The pCambia1303 plasmid is loaded into PMP by the method described in Example 5.

Encapsulation of the plasmid into the PMP is determined by PCR analysis. Whole genomic DNA is isolated from loaded PMP using standard methods. For PCR analysis, 50 ng of total DNA, 5 μl of RedTaq Readymix (Sigma Aldrich) and 0.1 μM each primer pair for amplifying the mgfp5 gene: 5'-AAG GAG AAG AAC TTT TCA CTG GAG-3' (SEQ ID NO: 7) and 5'-AGT TCA TCC ATG CCA TGT GTA-3 (SEQ ID NO: 8) are added to 10 μl of PCR. PCR amplification is a thermal cycler with initial denaturation at 95 ° C. for 1 minute followed by 30 cycles of 1 minute at 95 ° C., 1 minute at 55 ° C., 1 minute at 68 ° C. and 10 minutes at 68 ° C. Carry out within. PCR products were separated and imaged on a 1.0% agarose gel using electrophoresis. Gels are scored for the presence and intensity of the mgfp5 product (about 700 bp) compared to known amounts of the pCambia1303 plasmid. The plasmid-loaded PMP was formulated in water at concentrations delivering 0, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml plasmid equivalents in sterile water, etc. A volume concentration of unloaded PMP is used as a control. The stability of the plasmid load PMP is measured as described in Example 4.

Functional delivery of GUSA: mGFP5 reporter plasmid-loaded Grapefruit PMP in cotton plants Cotton seeds (Gossipium hilsutum and Gossypium raimondi) are the National Plant Genetic Resources System (US). Obtained through Germplasm System). Wrap sterilized seeds in hygroscopic cotton, place in Petri dishes, 14/10 hours (bright / dark) light with light intensity ^{of m-2} S- ^{1 at 25 ° C, 150 μE for 3 days to germinate.} Placed in the growth chamber under the period. Seedlings are grown in sterile culture vessels containing Hoagland nutrient solution (Sigma Aldrich) under long day conditions (16/8 hours light / dark period) using day / night temperatures of 26/20 ° C. After 4 days, seedlings with sufficiently large cotyledons (before the appearance of the first true leaf) are used for PMP treatment.

7-day-old cotton seedlings are 0.5 x Murashige scoog (MS) containing 1 x MS vitamin (Sigma Aldrich) (pH 5.6 to 5.8) containing 0.8% (w / v) agarose. ) By transferring onto an inorganic salt (Sigma Aldrich) and spraying 3 plants per group with a solution of 1 ml per plant on the whole seedling, the effective amount in sterile water (ddH2O) is 0, 0.5 μg. Treat with / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml plasmids and use equal concentrations of unloaded PMP as controls. Instead, prior to PMP treatment, the underside of the cotyledons of the cotton plant is perforated with a 25G needle without puncturing through the cotyledons. The PMP solution was formulated in (VIB medium (20 mM MES, 2 mM CaCl2 and 0.1 M NaCl, pH 6) for application underneath the leaflet through the wound site using a 1 mL needleless syringe. Manual infiltration from. Instead, 4-day-old seedlings are maintained under hydroponic growth conditions and a plasmid-loaded PMP formulation is added to the growth medium to determine root absorption by PMP. All plants grow. Transfer to chamber and maintain under long day conditions (16 hours / 8 hours light / dark period) with 90 μmol m- ² s- ^{1 light intensity and 26/20 ° C day / night temperature.}

After 1, 2, 3, 5, 8 and 14 days, the expression of GFP and GUS is determined to assess the functional delivery of plasmid loading PMP into plant cells. Total protein is extracted from 100 mg of fresh cotton leaves (method) and a vegetable protease inhibitor is added. Western blotting for GFP (Abcam ab1218) is performed using an actin (Abcam ab197345) loading control. Relative GFP expression normalized to actin is compared between plasmid loading PMP treatment, pharmaceuticals and controls. In addition, cotton plants are tested for GFP expression by fluorescence microscopy (EVOS2 FL) prior to protein isolation.

Histochemical localization of GUS activity in plasmid-loaded PMP-treated plants and controls was as follows: X-gluc buffer (50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0). The brightfield image is analyzed after incubation of the plant in 5 mM potassium ferrocyanide and 2 mg / ml 5-bromo-4-chloro-3-indrill glucuronide (X-gluc) at 37 ° C. for 12 hours. Collect using an anatomical microscope.

The pCambia1303 plasmid load grapefruit PMP functionally delivers their cargo to plant cells and successfully transcribes the reporter protein encoded in the plasmid.

Example 7: Modification of plants by PMP-mediated short-stranded nucleic acid delivery This example describes loading and functional delivery of short-stranded nucleic acid-loaded PMPs in plants to affect plant adaptability. This example further demonstrates that the short-stranded nucleic acid load PMP is stable over a range of processing and environmental conditions and retains their activity. In this example, cotton is used as a model plant, grapefruit PMP is used as a model PMP, and amiRNA for Cla1 is used as a model dsRNA.

Treatment plan:
Grapefruit PMP solution is formulated with 0 (negative control), 1, 5, 10 and 20 ng / μl dsRNA in sterile water.

Experimental protocol:
Loading CLA1 dsRNA into Grapefruit PMP An artificial targeting of the cotton photosynthetic gene GrCLA1 (1-deoxy-D-xylrose-5-phosphate synthase) in Grapefruit PMP to demonstrate cell absorption by PMP in plants. MiRNA (designed with amiRNA, P-SAMS (p-sama. Carringtonlab.org)) or custom dicer substrate siRNA (designed with DsiRNA, IDT) was loaded. GrCLA1 is a homologous gene of the Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) whose loss of function gives rise to an albino phenotype on the true leaf and provides a visual marker for silencing efficiency. Oligonucleotides are obtained from IDT.

PMP is produced from grapefruit as described in Example 1 and Example 2. To determine PMP uptake, the grapefruit PMP is loaded with a GrCLA1-amiRNA or GrCLA1-DsiRNA double helix (Table 1) as described in Example 5. AmiRNA or DsiRNA encapsulation of PMP is measured using the Quant-It RiboGreen RNA assay kit or a control fluorescent dye labeled with amiRNA or DsiRNA (IDT). To determine the PMP effect of CLA1-amiRNA / DsiRNA loaded PMP vs. non-loaded PMP, cotton seedlings are treated and analyzed for CLA1 gene silencing. PMP loaded with amiRNA or DsiRNA (collectively referred to as dsRNA) is formulated in water to deliver equivalent 0, 1, 5, 10 and 20 ng / μl effective dsRNA doses in sterile water. Equal concentrations of non-loaded PMP are delivered as controls. The stability of the dsRNA load PMP is measured as described in Example 4.

Functional delivery of grapefruit PMP loaded with dsRNA targeting CLA1 in cotton plants Cotton seeds (Gossipium hilsutum and Gossypium raimondi) are the US National Plant Genetic Resources System (USNation). Obtained through Plant Germplasm System). Sterilized seeds are wrapped in hygroscopic cotton, placed in a Petri dish and placed in a Petri dish for 3 days at 25 ° C. under 150 μE m- ² S- ¹ light intensity for 14/10 hours (light / dark) under light. Placed in the breeding chamber. Seedlings are grown in sterile culture vessels containing Hoagland nutrient solution (Sigma Aldrich) under long day conditions (16/8 hours light / dark period) using day / night temperatures of 26/20 ° C. After 4 days, seedlings with sufficiently large cotyledons (before the appearance of the first true leaf) are used for PMP treatment.

7-day-old cotton seedlings are 0.5 x Murashige scoog (MS) containing 1 x MS vitamin (Sigma Aldrich) (pH 5.6 to 5.8) containing 0.8% (w / v) agarose. Effective amounts of 0 (ddH2O), 1, 5, 10 and 20 ng / by transferring onto an inorganic salt (Sigma Aldrich) and spraying 3 plants per group with a solution of 1 ml per plant on the whole seedling. Treat with μl GrCLA1 dsRNA loaded PMP and unmodified grapefruit PMP with uniform PMP particle concentration. Instead, prior to PMP treatment, the underside of the cotyledons of the cotton plant is perforated with a 25G needle without puncturing through the cotyledons. The PMP solution is manually infiltrated from underneath the cotyledons through the wound site using a 1 mL needleless syringe. The plants are transferred to a growth chamber and maintained under long day conditions (16 hours / 8 hours light / dark period) with ^{90 μmol m-2} s- ^{1 light intensity and 26/20 ° C day / night temperature.} ..

After 2, 5, 8 and 14 days, the gene silencing efficiency of CLA1 dsRNA is tested by the expression level of endogenous CLA1 mRNA using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA is extracted from 100 mg of fresh cotton leaves using Trizol reagent according to the manufacturer's instructions (Invitrogen) and carefully treated with RNase-free DNase I (Promega). The first-strand cDNA is synthesized from 2 μg of total RNA using the SuperScript ™ First-Strand Synthesis System (Invitrogen). To estimate the level of the CLA1 transcript, qRT-PCR was performed in the following program: (a) 95 ° C for 5 minutes; (b) 94 ° C for 30 seconds, 55 ° C for 30 seconds; and 72 ° C for 30 seconds. Primers: GrCLA1q1_F 5'-CCAGGTGGGGCTTATGCATC-3'(SEQ ID NO: 9), GrCLA1q1_R 5'-CCACACCAAGGCTTGAACCC-3'(SEQ ID NO: 10) and GrCLA1q2_F 5'-GGCCG , GrCLA1q2_R 5'-CGTCGAGATTGGCAGTTGGC-3'(SEQ ID NO: 12) and 18s RNA_F 5'-TCTGCCCTATTCAACTTTCGAGGTA-3' (SEQ ID NO: 13), 18s RNA_R5'-AATTTGCGCT It is carried out using PCR Master Mix (Thermo Scientific). The 18S rRNA gene is used as an internal control to normalize the results. The CLA1 knockdown efficiency in cotton treated with CLA1-dsRNA loaded PMP compared to unmodified PMP and water control was that normalized CLA1 expression after treatment with dsRNA loaded PMP was normalized CLA1 expression after treatment with unmodified PMP. It is determined by calculating the ΔΔCt value by comparing with.

In addition, the gene silencing efficiency of CLA1 dsRNA is tested by phenotypic photobleaching analysis. Photographs of treated and untreated cotton plant leaves are taken and ImageJ software is used to determine the percentage of gene silencing reflected by white photobleaching on the leaves compared to the green color of the control leaves. Three leaves per plant are assayed to quantify the effect of photobleaching and the gene silencing efficiency of the CLA1-dsRNA loaded PMP pair control is evaluated.

CLA1-dsRNA loaded PMPs functionally deliver into plants and induce CLA1 gene silencing and photobleaching phenotypes as compared to unmodified PMPs and water controls.

Example 8: Modification of plants by PMP-mediated plasmid delivery to protoplasts This example describes loading and functional delivery of plasmid-loaded PMPs to protoplasts to affect plant fitness. This example also further demonstrates that the plasmid loaded PMP is stable and retains its activity over a variety of processing and environmental conditions.

In this example, soybean protoplasts are used as model protoplasts, BY-2 PMPs are used as model PMPs, and pCambia1303 GUSA: mGFP5 reporter expression vector is used as model plasmids.

Treatment plan:
The BY-2 PMP solution is formulated with equal doses of 0, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml plasmid solutions in sterile water.

Experimental protocol:
Loading the GUSA: mGFP5 reporter plasmid into the BY-2 PMP The PMP is generated from the BY-2 cell line as described in Examples 1 and 2. To demonstrate functional plasmid delivery by PMP, pCambia1303 reporter plasmid (12,361bp; Loaded by (purchased from Marker Gene Technologies). Upon transcription, both GUSA and mGFP5 are transcribed and can be detected. The pCambia1303 plasmid is loaded into PMP by the method described in Example 5.

Encapsulation of the plasmid into the PMP is determined by PCR analysis. Whole genomic DNA is isolated from loaded PMP using standard methods. For PCR analysis, 50 ng of total DNA, 5 μl of RedTaq Readymix (Sigma Aldrich) and 0.1 μM each primer pair for amplifying the mgfp5 gene: 5'-AAG GAG AAG AAC TTT TCA CTG GAG-3' (SEQ ID NO: 7) and 5'-AGT TCA TCC ATG CCA TGT GTA-3 (SEQ ID NO: 8) are added to 10 μl of PCR. PCR amplification is a thermal cycler with initial denaturation at 95 ° C. for 1 minute followed by 30 cycles of 1 minute at 95 ° C., 1 minute at 55 ° C., 1 minute at 68 ° C. and 10 minutes at 68 ° C. Carry out within. PCR products were separated and imaged on a 1.0% agarose gel using electrophoresis. Gels are scored for the presence and intensity of the mgfp5 product (about 700 bp) compared to known amounts of the pCambia1303 plasmid. The plasmid-loaded PMP was formulated in water to deliver 0, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml plasmids in sterile water and at equal concentrations. Non-loaded PMP is used as a control. The stability of the plasmid load PMP is measured as described in Example 4.

Isolation of Soybean Protoplasts Soybean seeds (Glycine max (L.) Merr.) Are obtained through the US National Plant Germplasm System. 5-10 soybean seeds (Williams 82), the custom soil mixture for soybean (1: 1: 1 ratio of soil, perlite and Topidosando) long-day conditions at 25 ° C. on (1,500μmolm ^-2 s ^- Planted in 13 cm pots in the growth chamber under (16 hours of illumination at ^1). Protoplasts are described by Wu and Hanzawa, 2018 J. Mol. Vis. Exp. (131), isolated as described in e57258. Briefly, newly elongated single-leaved leaves are cut from 10-day-old soybean seedlings. Using a new razor blade, remove the central vein from the leaves of the single leaf and cut the rest into 0.5-1 mm strips. Immediately transfer the leaf debris using a single forceps and 10 mL of freshly prepared enzyme solution (MES, pH 5.7, 20 mM, 2% (w / v) cellulase CELF, 0.1%) in a 15 mL tube. It was slowly placed in (w / v) pectrianase Y-23, 0.75 M mannitol, 0.2 mM CaCl2, 0.1% (w / v) BSA, 0.5 mM DTT). Leaf strips are vacuum infiltrated at room temperature for 15 minutes and these leaf strips are incubated in an enzyme solution at room temperature for 4-6 hours with gentle agitation under microlight (40 rpm). 10 mL of enzyme / protoplast solution is slowly infused into a 50 mL tube and 10 mL of W5 solution (2 mM MES, pH 5.7, 154 mM NaCl, 125 mM CaCl 2, 5 mM KCl) is added at room temperature to stop digestion. .. The enzyme / protoplast solution is poured onto a clean 75 μm nylon mesh placed on top of a 50 mL tube to remove undigested leaf tissue. The pass-through enzyme / protoplast solution is centrifuged at 100 xg in a 50 mL tube for 1-2 minutes at room temperature to remove the supernatant without interfering with the protoplast pellets. The resuspended protoplasts are diluted to a concentration ^{of 2 × 10 5} cells / ml in a cooled W5 solution at 4 ° C. by counting the number of protoplasts by blood cell counting. After washing, the protoplasts are resuspended in MMG solution (4 mM MES, pH 5.7, 400 mM mannitol, 15 mM MgCl2) at a concentration ^{of 2 × 10 5 cells / ml at room temperature.}

Functional delivery of BY-2PMP loaded with GUSA: mGFP5 reporter plasmid in soybean protoplasts Transfer 100 μL of protoplasts containing 2 × 10 ^{4 protoplasts at 2 × 10 5} cells / mL to a 24-well flat-bottomed plate. Protoplasts are treated with effective amounts of plasmids in 0, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml in sterile water (ddH2O) and are unloaded at equal concentrations. PMP is used as a control. If more than 100 ul of PMP solution needs to be added, PMP is formulated in MMG solution and inoculated 3 times per time point.

After 2 hours, 4 hours, 6 hours, 1 day, 2 days and 3 days, the expression of GFP and GUS is determined to evaluate the functional delivery of plasmid-loaded PMP into soybean protoplasts. Samples are washed with medium for 5 x 10 minutes as described above. All proteins are extracted from one whole well of the treated protoplast and Western blots for GFP (Abcam ab1218) are performed using an actin (Abcam ab197345) loading control. Relative GFP expression normalized to actin is compared between plasmid loading PMP treatment, pharmaceuticals and controls.

In addition, 10 μL of washed protoplasts are placed on glass slides and tested for GFP expression by fluorescence microscopy (EVOS2 FL).

Histochemical localization of GUS activity in plasmid-loaded PMP-treated protoplasts and controls was X-gluc buffer (50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0). The brightfield image is analyzed after incubation of protoplasts at 37 ° C. in 5 mM potassium ferrocyanide and 2 mg / ml 5-bromo-4-chloro-3-indrill glucuronide (X-gluc) for 12 hours. Obtained using EVOS2 FL.

The pCambia1303 plasmid load BY-2 PMP functionally delivers those cargoes to the protoplasts and successfully transcribes the reporter protein encoded in the plasmid.

Example 9: PMP generation from blended fruit juice using ultracentrifugation and sucrose gradient purification In this example, the fruits are blended, continuous centrifugation to remove debris, and pelletization of crude PMP. The production of PMP from fruit by using a combination of ultracentrifugation and using a sucrose density gradient for purifying PMP will be described. In this example, grapefruit was used as a model fruit.

a) Generation of grapefruit PMP by ultracentrifugation and sucrose density gradient purification A workflow for grapefruit PMP generation using blender, ultracentrifugation and sucrose gradient purification is shown in FIG. 1A. Purchase a red grapefruit from the local Whole Foods Market®, remove albedo, flaved and segmented membranes, collect juice vesicles and homogenize them using the fastest blender for 10 minutes. bottom. After diluting 100 mL of juice 5 times with PBS, large debris was removed by centrifugation at 1000 xg for 10 minutes, 3000 xg for 20 minutes and 10,000 xg for 40 minutes. 28 mL of clarified juice was ultracentrifuged for 90 minutes in a 150,000 xg Sorvall ™ MX 120 Plus Micro-Ultracentrifuge at 4 ° C using an S50-ST (4 x 7 mL) swing bucket rotor. , Crude PMP pellets were obtained and resuspended in PBS pH 7.4. Next, a sucrose gradient is prepared in Tris-HCL pH 7.2 and a crude PMP is layered on top of the sucrose gradient (top to bottom: 8, 15, 30, 45 and 60% sucrose). Using an S50-ST (4 × 7 mL) swing bucket rotor, spin-down was performed by ultracentrifugation at 4 ° C. and 150,000 × g for 120 minutes. 1 mL fraction was collected and PMP was isolated at the 30-45% interface. Fractions were washed with PBS by ultracentrifugation at 4 ° C. at 150,000 xg for 120 minutes and the pellet was dissolved in a minimum amount of PBS.

^{A PMP concentration (1 × 10 9} PMP / mL) and a median PMP particle size (121.8 nm) were determined using a Specradine nCS1 ™ particle size analyzer using a TS-400 cartridge (FIG. 1B). The zeta potential was measured using the Malvern Zetasizer Ultra and found to be -11.5 +/- 0.357 mV.

This example demonstrates that grapefruit PMP can be isolated using ultracentrifugation in combination with the sucrose gradient purification method. However, this method induced significant gelation of the sample in all PMP generation steps and in the final PMP solution.

Example 10: PMP generation from mesh pressed fruit juice using ultracentrifugation and sucrose gradient purification This example is a cell wall during the PMP generation step by using a milder juice method (mesh strainer). And the reduction of cell membrane contaminants will be explained. In this example, grapefruit was used as a model fruit.

a) Gently squeezing reduces gelation during PMP production from grapefruit PMP. Juice vesicles were isolated from red grapefruit as described in Example 9. To reduce gelation during PMP formation, instead of using a destructive blending method, a juice vesicle was lightly pressed against the tea strainer mesh to collect juice and reduce cell wall and cell membrane contaminants. After fractional centrifugation, the juice became clearer than after the use of Brenda, and one pure PMP-containing sucrose band was observed at the 30-45% intersection after sucrose density gradient centrifugation (FIG. 2). Overall, there was little gelation during and after PMP formation.

Our data demonstrate that the use of a gentle squeezing step reduces gelation due to contaminants during PMP production compared to methods involving blending.

Example 11: PMP generation using ultracentrifugation and size exclusion chromatography This example describes the production of PMP from fruits by using ultracentrifugation (UC) and size exclusion chromatography (SEC). In this example, grapefruit is used as a model fruit.

a) Generation of grapefruit PMP using UC and SEC As described in Example 9a, juice vesicles were isolated from red grapefruit and lightly pressed against a tea strainer mesh to collect 28 ml of juice. The workflow of grapefruit PMP generation using UC and SEC is depicted in FIG. 3A. Briefly, large debris was removed by subjecting the juice to fractional centrifugation at 1000 xg for 10 minutes, 3000 xg for 20 minutes and 10,000 xg for 40 minutes. 28 ml of clarified fruit juice was ultracentrifuged at 4 ° C. using a S50-ST (4 x 7 mL) swing bucket rotor with 100,000 x g Sorvall ™ MX 120 Plus Micro-Ultracentrifuge for 60 minutes. , Crude PMP pellets were obtained and resuspended in MES buffer (20 mM MES, NaCl, pH 6). After washing the pellet twice with MES buffer, the final pellet was resuspended in 1 ml PBS, pH 7.4. The PMP-containing fraction was then eluted using size exclusion chromatography. The SEC elution fraction was analyzed by nano-flow cytometry with NanoFCM to determine the PMP particle size and concentration using the concentration and particle size standards provided by the manufacturer. In addition, the absorbance at 280 nm (SpectraMax®) and protein concentration (Pierce ™ BCA assay, Thermo Fisher) were determined for the SEC fraction to identify in which fraction the PMP was eluted. (FIGS. 3B to 3D). SEC fractions 2-4 were identified as PMP-containing fractions. Analysis of the early and late elution fractions revealed that the SEC fraction 3 was the major PMP-containing fraction, with a concentration of 2.83 × 10 ¹¹ PMP / mL (57.2% of the total particles was 50). The median diameter was 83.6 nm +/- 14.2 nm (SD) (in the ~ 120 nm particle size range). Late-eluting fractions 8-13 had very low concentrations of particles, as shown by NanoFCM, but protein contaminants were detected in these fractions by BCA analysis.

The data of the present inventors are that the purified PMP can be isolated from the late-eluting contaminants using TFF and SEC, and the combination of the analytical methods used in this example allows the PMP image from the late-eluting contaminants. It proves that the minute can be identified.

Example 12: Scaled PMP generation using tangential flow filtration and size exclusion chromatography in combination with EDTA / dialysis to reduce contaminants This example is an EDTA incubation to reduce the formation of pectin macromolecules. And explained the scaled production of PMP from fruit by using tangential flow filtration (TFF) and size exclusion chromatography (SEC) in combination with overnight dialysis to reduce contaminants. do. In this example, grapefruit is used as a model fruit.

a) Generation of grapefruit PMP using TFF and SEC Red grapefruit was purchased from the local Whole Foods Market® and 1000 ml of juice was isolated on a juice squeezer. The workflow for the generation of grapefruit PMP using TFF and SEC is depicted in FIG. 4A. Large debris was removed by subjecting the juice to fractional centrifugation at 1000 xg for 10 minutes, 3000 xg for 20 minutes and 10,000 xg for 40 minutes. The clarified grapefruit juice was concentrated and washed once to 2 mL (100 ×) using TFF (pore size 5 nm). The PMP-containing fraction was then eluted using size exclusion chromatography. The SEC elution fraction was analyzed by nano-flow cytometry with NanoFCM to determine the PMP concentration using the concentration and particle size standards provided by the manufacturer. Furthermore, in order to identify in which fraction the PMP was eluted, the protein concentration (Pierce ™ BCA assay, Thermo Fisher) was determined for the SEC fraction. In scaled production from 1 liter of juice (100-fold concentrated), the BCA assay can detect large amounts of contaminants that are also concentrated in the late SEC fraction (FIG. 4B, upper panel). Overall total PMP yield (FIG. 4B, lower panel) was lower for scaled production compared to single grapefruit isolation, which may indicate a loss of PMP.

b) Reduction of contaminants by EDTA incubation and dialysis Red grapefruit was purchased from the local Whole Foods Market® and 800 ml of juice was isolated using a juice squeezer. After removing large debris by subjecting the juice to fractional centrifugation at 1000 xg for 10 minutes, 3000 xg for 20 minutes and 10,000 xg for 40 minutes, with a 1 μm and 0.45 μm filter. Filtration was performed to remove large particles. The clarified grapefruit juice was divided into four different treatment groups, each containing 125 ml of juice. The treatment group 1 was treated as described in Example 12a, concentrated and washed (PBS) to a final concentration of 63 ×, and subjected to SEC. To chelate iron prior to TFF and reduce the formation of pectin macromolecules, 475 ml of juice was incubated at RT for 1.5 hr with a final concentration of 50 mM EDTA, pH 7.15. The juices were then divided into three treatment groups and subjected to TFF concentration to a final juice concentration of 63x with either PBS (calcium / magnesium free) pH 7.4, MES pH 6 or Tris pH 8.6 wash solution. .. The sample was then dialyzed overnight at 4 ° C. in the same wash buffer using a 300 kDa membrane and then subjected to SEC. High contaminants in the control's late elution fraction of TFF alone, as shown by absorbance at 280 nm (FIG. 4C) and BCA protein analysis (which is sensitive to the presence of sugars and pectin) (FIG. 4D). Overnight dialysis after EDTA incubation compared to peak significantly reduced contaminants. There was no difference in the dialysis buffers used (PBS pH 7.4, MES pH 6, Tris pH 8.6 without calcium / magnesium).

Our data show that EDTA incubation followed by dialysis reduces the amount of co-purified contaminants and promotes scaled PMP production.

Example 13: PMP Generation from Plant Cell Medium This example illustrates the production of PMP from plant cell medium. In this example, a Zea mays Black Mexican sweet (BMS) cell line is used as a model plant cell line.

a) Generation of Zea mays BMS cell line PMP A Black Mexican sweet (BMS) cell line was purchased from ABRC and 4.3 g at 24 ° C. / L Murashige and Skoog base salt mixture (Sigma M5524), 2% sucrose (S0389, Millipore Sigma), 1 × MS vitamin solution (M3900, Millipore Sigma), 2 mg / L 2,4-dichlorophenoxyacetic acid (D7299, Millipore) Growing in Murashige and Skoog base medium pH 5.8 containing Sigma) and 250 ug / L of thiamine HCL (V-014, Millipore Sigma) with stirring (110 rpm) and subcultured at 20% volume / volume every 7 days. It was cultured.

Three days after passage, 160 ml of BMS cells were collected and spun down at 500 xg for 5 minutes to remove the cells, followed by a spin down at 10,000 xg to remove large debris. The medium was passed through a 0.45 μm filter to remove large particles, and the filtered medium was concentrated and washed (100 ml MES buffer, 20 mM MES, 100 mM NaCl, pH 6) to 4 mL (40 ×) with TFF (pore size 5 nm). The PMP-containing fraction was then eluted using size exclusion chromatography and analyzed by absorbance at 280 nm (SpectraMax®) and protein concentration assay (Pierce ™ BCA assay, Thermo Fisher). Then, the PMP-containing fraction and the late fraction containing the contaminants were confirmed (FIGS. 5A to 5C). SEC fractions 4-6 contained purified PMP (fractions 9-13 contained contaminants), which were pooled together. ^{The final PMP concentration (2.84 × 10 10} PMP / ml) and median PMP particle size (63.2 nm +/- 12) of the combined PMP-containing fractions by NanoFCM using the concentration and particle size standards provided by the manufacturer. .3 nm SD) was determined (FIGS. 5D-5E).

These data indicate that PMP can be isolated, purified and concentrated from plant liquid media.

Example 14: Uptake of PMP by Plant Cells In this example, the ability of PMP to bind to and be taken up by plant cells is described. In this example, lemon PMP is used as the model PMP and soybean, wheat and corn cell lines are used as the model plant cells.

a) Generation of Grapefruit PMP Using TFF in Combination with SEC Red Organic Grapefruit (Florida) was obtained from the local Whole Foods Market®. Large debris was removed by collecting 1 liter of grapefruit juice using a juice squeezer and then centrifuging at 3000 xg for 20 minutes followed by 10,000 xg for 40 minutes. Next, 500 mM EDTA (pH 8.6) was added to a final concentration of 50 mM EDTA (pH 7) to chelate calcium and incubate the solution for 30 minutes to prevent the formation of pectin macromolecules. Subsequently, the juice was passed through 11 μm, 1 μm and 0.45 μm filters to remove large particles. The filtered fruit juice is concentrated and washed (500 ml PBS) by tangential flow filtration (TFF) (pore size 5 nm) to 400 ml (2.5 ×), and then 1 in PBS (pH 7.4) using a 300 kDa dialysis membrane. The contaminants were removed by late dialysis (including one media change). Then, the dialyzed juice was concentrated to a final concentration (20 ×) of 50 ml by TFF. The PMP-containing fraction was then eluted using size exclusion chromatography and analyzed by 280 nm absorbance (SpectraMax®) and protein concentration assay (Pierce ™ BCA assay, Thermo Fisher). , PMP-containing fraction and late fraction containing contaminants were confirmed. SEC fractions 4-6 contain purified PMP (fractions 8-14 contain contaminants), which are pooled together and 0.8 μm, 0.45 μm and 0.22 μm syringes. It was sterilized by filtration by continuous filtration using a filter. ^{Final PMP concentration (1.32 × 10 11} PMP / mL) and median PMP particle size (71.9 nm +/-) of combined sterile PMP-containing fractions by NanoFCM using the concentration and particle size standards provided by the manufacturer. 14.5 nm) was determined.

b) Lemon PMP production using TFF in combination with SEC Lemons were obtained from the local Whole Foods Market®. Large debris was removed by collecting 1 liter of lemon juice using a juice squeezer and then centrifuging at 3000 g for 20 minutes followed by 10,000 g for 40 minutes. Next, 500 mM EDTA (pH 8.6) was added to a final concentration of 50 mM EDTA (pH 7) to chelate calcium and incubate the solution for 30 minutes to prevent the formation of pectin macromolecules. Subsequently, the juice was passed through a coffee filter, 1 μm and 0.45 μm filters to remove large particles. The filtered fruit juice is concentrated to 400 ml (2.5 x concentrated) by tangential flow filtration (TFF) (pore size 5 nm), then dialyzed overnight in PBS (pH 7.4) using a 300 kDa dialysis membrane and mixed. The thing was removed. Then, the dialyzed juice was concentrated to a final concentration (20 ×) of 50 ml by TFF. The PMP-containing fraction was then eluted using size exclusion chromatography and analyzed by 280 nm absorbance (SpectraMax®) and protein concentration assay (Pierce ™ BCA assay, Thermo Fisher). , PMP-containing fraction and late fraction containing contaminants were confirmed. SEC fractions 4-6 contain purified PMP (fractions 8-14 contain contaminants), which are pooled together and 0.8 μm, 0.45 μm and 0.22 μm syringes. It was sterilized by filtration by continuous filtration using a filter. ^{The final PMP concentration (2.7 × 10 11} PMP / mL) and median PMP particle size (70.7 nm +/-) of the combined sterile PMP-containing fractions by NanoFCM using the concentration and particle size standards provided by the manufacturer. 15.8 nm) was determined.

c) Labeling of Lemon PMP with Alexa Fluor 488 NHS Ester Lemon PMP was produced as described in Example 14b. PMP was labeled with Alexa Fluor 488® NHS Ester (Life Technologies, Covalent Bond Membrane Dye (AF488)). Briefly, AF488 is dissolved in DMSO to a final concentration of 10 mg / ml, 200 ul of PMP (1.53E + 13 PMP / ml) is mixed with 5 ul of dye, incubated for 1 h on a shaker at room temperature, and then 1 hr at 4 ° C. The labeled PMP was washed 2-3 times by ultracentrifugation of 100,000 xg. The pellet was resuspended in 1.5 ml of UltraPure water. To verify the presence of potential dye aggregates, 200 ul of UltraPure water was added in place of PMP and a control sample of dye alone was prepared according to the same procedure. The final AF488-labeled PMP pellet and AF488 dye-only control were resuspended in a minimum amount of UltraPure water and characterized by NanoFCM. The final concentration of lemon 488-labeled PMP was 2.91 × 10 ¹² PMP / ml, the median AF488-PMP particle size was 79.4 nm +/- 14.7 nm, and the labeling efficiency was 89.4% ( FIG. 6A).

d) Incorporation of AF488-labeled lemon PMP by plant cells The plant cell line was derived from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Glycine max, # PC-1026; Triticham estivam). 998) and ABRC (Zea mays, Black Mexican sweets (BMS), agitated (110 rpm) in a vented 250 mL flask with a baffle in the dark at 24 ° C. Glycine max and Triticum aestivum were grown with 2% sucrose and 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4D) according to the supplier's instructions. (D7299, Millipore Sigma) was added and grown on a 3.2 g / L Gamborg B-5 basal medium (Gamborg's B-5 Basal Medium) containing Minimal Organics supplement (G5893, Millipore Sigma) pH 5.5. BMS cells are 4.3 g / L Murashige scoog base salt mixture (Sigma M5524), 2% sucrose (S0389, Millipore Sigma), 1 × MS vitamin solution (M3900, Millipore Sigma), 2 mg / L 2,4. -They were grown in Murashige scoog medium pH 5.8 containing dichlorophenoxyacetic acid (D7299, Millipore Sigma) and 250 ug / L thiamine HCL (V-014, Millipore Sigma).

For treatment with AF488-PMP, 5 mL of cell suspension was harvested to determine% pack cell volume (PCV). PCV is defined as the quotient of cell volume divided by the total volume of cell culture aliquots and is expressed as a percentage. The cells were centrifuged at 3900 rpm for 5 minutes to determine the volume of cell pellets. The% PCVs of BMS, Glycine max and Triticum aestivum were 20%, 15% and 18%, respectively. For uptake experiments, the% PCV of the culture was adjusted to 2% by diluting the cells with its appropriate medium. Next, 125 μl of plant cell suspension was added to the 24-well plate and the double sample was added to 125 μl MES buffer (200 mM MES + 10 mM NaCl, pH 6) alone (negative control), AF488 dye alone (dye alone control) or 125 μl. Treated to the final concentration of 1 × 10 ¹² AF488-PMP / mL diluted with MES buffer. After incubating the cells in the dark at 24 ° C. for 2 hours and washing 3 times with 1 mL of MES buffer to remove the AF488-PMP or free dye that was not taken up, an epifluorescence microscope (EVOS FL Auto 2, Invitrogen). Resuspended in 300 μL MES buffer for imaging in. Various fluorescence signals indicating PMP uptake could be detected in all plant cell lines as compared to the AF488 dye-only control, which had no detectable fluorescence (FIG. 6B). Triticum aestivum cells exhibited the strongest fluorescent signal, indicating that of the three plant cell lines tested, they had the highest uptake of AF488-labeled lemon PMP.

Our data show that PMP can be taken up in vitro by plant cells.

Example 15: Uptake of PMP in Plants This example describes uptake and systemic transport of PMP in plants. In this example, grapefruit, lemon and Arabidopsis thaliana seedling PMP are used as model PMPs, and Arabidopsis seedlings and alfalfa sprout are used as model plants.

a) Labeling of Lemon and Grapefruit PMP with DyLight 800 NHS Ester Grapefruit and lemon PMP were produced as described in Examples 14a and 14b. PMP was labeled with DyLight 800 NHS Ester (Life Technologies, # 46421) covalent membrane dye (DyL800). Briefly, Dyl800 is dissolved in DMSO to a final concentration of 10 mg / ml, then 200 μl of PMP is mixed with 5 μl of dye, incubated for 1 h on a shaker at room temperature, and then 1 hr at 4 ° C., 100,000 × g. The labeled PMP was washed 2-3 times by ultracentrifugation and the pellet was resuspended in 1.5 ml UltraPure water. To verify the presence of potential dye aggregates, 200 μl UltraPure water was added in place of PMP and a control sample of dye alone was prepared according to the same procedure. The final DyL800-labeled PMP pellet and DyL800 dye-only control were resuspended in a minimum amount of UltraPure water and characterized by NanoFCM. The final concentration of grapefruit DyL800 labeled PMP was 4.44 × 10 ¹² PMP / ml and the final concentration of lemon DyL800 labeled PMP was 5.18 × 10 ¹² PMP / ml. Labeling efficiency could not be determined using NanoFCM because NanoFCM cannot detect infrared light.

b) Arabidopsis thaliana seedling germination and growth Wild-type Arabidopsis thaliana Col-0 seeds are obtained from ABRC, surface sterilized with 70% ethanol, and then surface sterilized with 50% bleach / 0.1% Triton X. -100 and was incubated for 10 minutes together by four sterile ddH ₂ O wash to remove the bleach solution. Seeds were stratified over 1d in the dark at 4 ° C. ^{Per 100 cm 2} plate (pre-coated with 0.5% fetal bovine serum in water) containing 20 mL of 0.5 x MS medium (2.15 g / L Murashige and Skoug salt, 1% sucrose, pH 5.8). Approximately 250 seeds were germinated, sealed with 3M surgical tape and grown in an incubator using a light cycle of 23 ° C. for 16 h light and 21 ° C. for 8 h dark.

c) Incorporation of DyL800-labeled grapefruit, lemon and At PMP by Arabidopsis thaliana and Alfalfa The top of the mesh filter to determine if PMP can be taken up and systematically transported by the implanter. In addition, Arabidopsis seedlings are germinated in a liquid medium as described in Example 15b to allow roots to grow through the mesh, allowing partial exposure of At seedlings to PMP solution. Alfalfa sprout was acquired from a local supermarket. 9-day-old Arabidopsis seedlings and alfalfa sprout in 0.5 x MS medium water (negative control), DyL800 dye alone (dye control), DyL800 labeled grapefruit PMP (1.6 x 10 ¹⁰ PMP / ml) ) Or a 0.5 ml solution of lemon (5.1 x 10 ¹⁰ PMP / ml), partial root exposure (to seedlings in a mesh floating in a PMP solution or 1.5 ml in an Eppendorf tube in alfalfa sprout). (By root exposure) was treated at 23 ° C. for 22 or 24 hours, respectively. The plants were then washed 3 times with MS medium and then imaged with Odyssey® CLx infrared imager (Li-Cor).

All PMP sources showed fluorescence signals in both Arabidopsis seedlings and alfalfa sprout (white is high fluorescence) compared to negative (some autofluorescence on alfalfa sprout leaves) and dye-only controls. It is a signal, and black has no signal) (Fig. 7). The presence of fluorescent signals in Arabidopsis leaves or alfalfa stem regions that were not exposed to PMP solution indicates active transport of PMP in the implanter. The DyL800 treatment concentration was not normalized in this experiment and therefore the difference in source / target uptake efficiency cannot be evaluated.

Our data show that PMPs from various plant sources can be taken up and transported by implanters.

Example 16: Treatment of Arabidopsis thaliana seedlings with DOX Road Grapefruit PMP This example illustrates the loading of small molecules into PMP for the purpose of reducing plant fitness. In this example, doxorubicin is used as a small molecule and Arabidopsis thaliana is used as a model plant. Doxorubicin is described in Streptomyces peusetius var. It is a cytotoxic anthracycline antibiotic isolated from a culture of Streptomyces peuceticus var. Caesius. Doxorubicin interacts with DNA by intercalation and inhibits both DNA replication and RNA transcription. Doxorubicin has been shown to be cytotoxic in plants (Culiarez-Mac et al, Plant Growth Regulation, (5): 155-164, 1987).

Effective and safe herbicides are needed to prevent significant crop yield losses due to weeds while protecting the environment from the toxic side effects of overuse of herbicides.

a) Generation of grapefruit PMP using TFF in combination with SEC Red organic grapefruit was obtained from the local Whole Foods Market®. 4 liters of grapefruit juice is collected using a juice squeezer, pH adjusted to pH 4 with NaOH, incubated with 1 U / ml pectinase (Sigma, 17389) to remove pectin contaminants, and then 3, Large debris was removed by centrifugation at 000 g for 20 minutes followed by 10,000 g for 40 minutes. The treated juice is then incubated with 500 mM EDTA (pH 8.6) (up to a final concentration of 50 mM EDTA (pH 7.7)) for 30 minutes to chelate calcium and prevent the formation of pectin macromolecules. bottom. Subsequently, the EDTA-treated juice was passed through 11 μm, 1 μm and 0.45 μm filters to remove large particles. The filtered juice was washed and concentrated by tangential flow filtration (TFF) using a 300 kDa TFF. After concentrating the juice 5 ×, 6 volume exchange washes with PBS were performed and further filtered to a final concentration of 198 mL (20 ×). Next, size exclusion chromatography was used to elute the PMP-containing fraction, which was analyzed by absorbance at 280 nm (SpectraMax®) and protein concentration (Pierce ™ BCA assay, Thermo Fisher). , PMP-containing fraction and late fraction containing contaminants were confirmed. SEC fractions 3-7 contained purified PMP (fractions 9-12 contained contaminants), which were pooled together to 0.8 μm, 0.45 μm and 0. After filtration sterilization by continuous filtration using a 22 μm syringe filter, PMP is pelleted at 40,000 × g for 1.5 hours and pelleted in 4 ml UltraPure ™ DNase / RNase-free distilled water (Thermo Fisher, 10977023). It was further concentrated by resuspending. ^{The final PMP concentration (7.56 × 10 12} PMP / ml) and average PMP particle size (70.3 nm +/- 12.4 nm (SD)) were obtained by NanoFCM using the concentration and particle size standards provided by the manufacturer. Decided. The produced grapefruit PMP was used to load doxorubicin.

b) Loading of doxorubicin into grapefruit PMP The grapefruit PMP produced in Example 16a was used for loading doxorubicin (DOX). A stock solution of doxorubicin (Sigma PHR1789) was prepared at a concentration of 10 mg / mL in UltraPure water and sterilized by filtration (0.22 μm). Sterile grapefruit PMP (3 mL at a particle concentration of 7.56 × 10 ¹² PMP / ml) was mixed with 1.29 mL of DOX solution. The final DOX concentration in the mixture was 3 mg / mL. The mixture was sonicated in an ultrasonic bath (Branson 2800) for 20 minutes while warming to 40 ° C. and then maintained in a bath without ultrasound for an additional 15 minutes. The mixture was stirred at 24 ° C. and 100 rpm for 4 hours in the dark. The mixture was then extruded using an Avanti Mini Extruder (Avanti Lipids). DOX-loaded PMPs were extruded at tapering methods: 800 nm, 400 nm and 200 nm to reduce the number of lipid bilayers and overall particle size. The extruded sample was sterilized by filtration by sequentially passing it through a 0.8 μm and 0.45 μm filter (Millipore, diameter 13 mm) in a TC hood. Samples were purified using an ultracentrifugation approach to remove non-loaded or weakly bound DOX. Specifically, it was spun down at 100,000 × g at 4 ° C. for 1 hour in a 1.5 mL ultracentrifuge tube. The supernatant was collected for further analysis and stored at 4 ° C. The pellet was resuspended in sterile water and ultracentrifuged under the same conditions. This step was repeated 4 times. The final pellet was resuspended in sterile UltraPure water and then stored at 4 ° C. until the next use.

Next, the concentration of particles and the loading capacity of PMP were determined. NanoFCM was used to determine the total number of PMPs in the sample (4.76 × 10 ¹² PMP / ml) and median particle size (72.8 nm +/- 21 nm (SD)). The DOX concentration was evaluated by fluorescence intensity measurement (Ex / Em = 485/550 nm) using a SpectraMax® spectrophotometer. Calibration curves for 0-50 μg / mL free DOX were made in sterile water. To dissociate the DOX-loaded PMP and DOX complex (π-π stacking), samples and standards were incubated with 1% SDS for 45 minutes at 37 ° C. prior to fluorescence measurements. The loading capacity (Pg of DOX per 1000 particles) was calculated as the quotient of the concentration of DOX (pg / ml) divided by the total number of PMPs (PMP / ml). The PMP-DOX loading capacity was 1.2 pg DOX per 1000 PMP. However, it should be noted that the load efficiency (% of the DOX load PMP relative to the total number of PMPs) could not be evaluated because the DOX fluorescence spectrum could not be detected by NanoFCM.

c) Treatment of Arabidopsis thaliana seedlings with doxorubicin-loaded PMP Grapefruit PMP was produced and loaded with doxorubicin as described in Examples 16a and 16b. Wild Arabidopsis thaliana Col-0 seeds were obtained from ABRC, surface sterilized with 50% bleaching agent, stratified at 4 ° C for 1-3d, and then containing 0.8% agar. 1/2 concentration (0.5 ×) Murashige and Skoog (MS) medium supplemented with 0.5% sucrose, 2.5 mM MES, pH 5.6, 16 h light period at 23 ° C / 8 h dark period light at 21 ° C. It was germinated using a cycle.

To test whether PMP can deliver small molecule cargo in an implanter, 7-day-old Arabidopsis thaliana seedlings were placed in a 24-well plate (1 seedling per well) 0.5 × It was transferred to liquid MS medium and then treated with 0 (negative control), 25 μM, 50 μM and 100 μM encapsulated DOX doses of free DOX or DOX-loaded PMP. The plates were covered with aluminum foil and incubated for 24 hours. The DOX-containing medium was removed, the seedlings were washed twice with 1/2 × MS medium, and then fresh medium was added. Seedlings were incubated for an additional 3 days under a normal photoperiod (16 h light period 23 ° C / 8 h dark period 21 ° C). The seedlings were then removed from the plate, dried with a towel for imaging, and then evaluated for cytotoxicity by analyzing leaf vitality, leaf color and root length. Cytotoxicity is defined by root shortening, leaf vitality loss and leaf fading (yellow rather than green) compared to untreated seedling controls. PMPs loaded with DOX were cytotoxic at 50 μM and 100 μM DOX, as compared to free DOX, which showed cytotoxicity (root shortening and leaf fading) only at 100 μM DOX. Seedlings treated with 50 μM PMP-DOX showed marked leaf yellowing with reduced leaf vitality and root shortening. Our data show that PMP can be loaded with small molecules in an implanter and deliver small molecules, and that PMP loaded with doxorubicin induces cytotoxic responses twice as efficiently as free doxorubicin. It is shown that.

Example 17. Incorporation of pectinase-treated PMP with alfalfa sprout This example describes that removal of pectin during the PMP production process does not impact their implanter uptake and systemic transport. In this example, lemon PMP was used as the model PMP and alfalfa sprout was used as the model plant.

a) Lemon PMP production with or without pectinase Lemons were obtained from the local Whole Foods Market®. Lemon juice (1260 ml) was collected using a juice squeezer and divided into two fractions. 630 ml was untreated and 630 ml was pH adjusted to pH 4 with NaOH and incubated with 6 U / ml pectinase (Sigma, 17389) for 1.45 hours at room temperature. Large debris was removed by centrifuging the pectinase-treated and untreated juice at 3000 g for 20 minutes and then at 10,000 g for 40 minutes. The treated juice is then combined with 500 mM DTA (pH 8.6) (final concentration 50 mM EDTA (pH 7.19-7.25)) to chelate calcium and prevent the formation of pectin macromolecules. Was incubated at room temperature for 30 minutes. Subsequently, the EDTA-treated juice was passed through 11um, 1um and 0.45um filters to remove large particles. The filtered juice was washed (260 ml PBS during the TFF process), concentrated about 1.6 × to a total volume of 400 ml by tangential flow filtration (TFF), and then dialyzed overnight in PBS using a 300 kDa dialysis membrane. Then, the dialyzed juice was further concentrated by TFF to a final concentration of 30 ml (about 21 ×). Next, size exclusion chromatography is used to elute the PMP-containing fraction and analyze the absorbance at 280 nm (SpectraMax) to determine the PMP-containing fraction from the late elution fraction containing contaminants. SEC fractions 4-6 (without pectinase treatment) and SEC fractions 4-7 (with pectinase treatment) containing purified PMP were pooled together in the individual treatment group. Pooled SEC fractions were dialyzed overnight in PBS (pH 7.4) using a 300 kDa dialysis membrane. Samples are sterilized by continuous filtration using 0.85 um, 0.4 um and 0.22 um syringe filters and further concentrated by pelleting PMP at 40,000 xg for 1.5 hours and finally pelleting. Is resuspended in ultrapure water. Using the concentration and particle size standards provided by the manufacturer, the final PMP concentration for untreated lemon PMP is 1.24 × 10 ¹² PMP / ml, as determined by nanoflow cytometry (NanoFCM). The median PMP particle size was 129 nm +/- 12 nm (SD); for the pectinase-treated lemon PMP, the final concentration was 2.2610 ¹² PMP / ml and the median PMP particle size was 130 nm +/- 11 nm (SD). )Met.

b) Labeling of Lemon PMP with DyLight 800 NHS Ester Pectinase-treated and untreated lemon PMP was labeled with DyLight 800 NHS Ester (Life Technologies, # 46421) covalent bond membrane dye (DyL800). Briefly, DyL800 is dissolved in DMSO to a final concentration of 10 mg / ml, 200 ul of PMP is mixed with 5 ul of dye, incubated for 1 hour on a shaker at room temperature and then 100,000 × for 1 hour at 4 ° C. After washing the labeled PMP 2-3 times by ultra-centrifugation of g, the pellet was resuspended in 1.5 ml of ultrapure water. To verify the presence of potential dye aggregates, 200 ul of UltraPure water was added in place of PMP and a control sample of dye alone was prepared according to the same procedure. The final DyL800-labeled PMP pellet and DyL800 dye-only control were resuspended in a minimum amount of UltraPure water and characterized by NanoFCM. The final concentration of non-pectinase-treated Dyl800-labeled lemon PMP was 3.2 × 10 ¹² PMP / ml, and the final concentration of pectinase-treated DyL800-labeled was 5.57 × 10 ¹² PMP / ml. Labeling efficiency could not be determined using nanoFCM because NanoFCM cannot detect infrared light.

c) Treatment of alfalfa sprout with pectinase-treated and untreated DyL800-PMP To assess whether removal of pectin during PMP production affects PMP uptake, alfalfa sprout is halved. Obtained from a local supermarket supplemented with 0.5% sucrose in MS) and 2.5 mM MES (pH 5.6), pectinase-treated and untreated DyLight 800 Lemon PMP, water (negative control), DyLight 800 nm dye. Treatment alone (dye aggregate control) at 23 ° C. for 21 hours (FIG. 8A). The seedlings were then washed 3 times in MS medium and imaged using an Odyssey infrared imager. No difference was found in the uptake and transport of PMP produced with or without pectinase treatment (FIG. 8B).

Example 18: Modification of PMP with Cationic Lipids In this example, modification of PMP with cationic lipids modifies surface charge, increases cargo loading capacity, and of PMP in plant cells. Prove the ability to increase cell uptake. In this example, DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DC-cholesterol (3β- [N- (N', N'-dimethylaminomethane) -carbamoyl] cholesterol) are model cationic. As lipids, Grapefruit and Lemon PMP as model PMPs, siRNA / trans-activated CRISPR RNA (TracrRNA) as model loading electroloading and Zea mays Black Mexican sweet (BMS). Used as a model plant cell line.

Experimental protocol:
a) Lemon / Grapefruit PMP Generation Red Organic Grapefruit or Yellow Organic Lemon was obtained from a local grocery store. 6 liters of grapefruit juice was collected using a juice squeezer, pH adjusted to pH 4 with NaOH, incubated with 1 U / ml pectinase (Sigma, 17389) to remove pectin contaminants, and then 3, Large debris was removed by centrifugation at 000 g for 20 minutes followed by 10,000 g for 40 minutes. The treated juice is then incubated with 500 mM EDTA (pH 8.6) (up to a final concentration of 50 mM EDTA (pH 7.7)) for 30 minutes to chelate calcium and prevent the formation of pectin macromolecules. bottom. Subsequently, the EDTA-treated juice was passed through 11 μm, 1 μm and 0.45 μm filters to remove large particles. The filtered juice was washed and concentrated by tangential flow filtration (TFF) using a 300 kDa TFF. After the juice was concentrated 10 ×, dialysis filtration was performed on 10 diavolumes in PBS and further concentrated to a final concentration of 120 mL (50 ×). The PMP-containing fraction was then eluted using size exclusion chromatography (SEC) and analyzed by absorbance at 280 (SpectraMax®) and protein concentration (Pierce® BCA Protein Assay). Then, the PMP-containing fraction and the late fraction containing the contaminants were confirmed. SEC fractions 3-7 contain purified PMP (fractions 9-12 contain contaminants), which are pooled together and 0.8 μm, 0.45 μm and 0.22 μm syringes. After filtration sterilization by continuous filtration using a filter, PMP is pelleted at 40,000 xg for 1.5 hours and the pellet is resuspended in 4 mL UltraPure ™ DNase / RNase-free distilled water (Thermo Fisher, 10977023). It was further concentrated by making it turbid. ^{The final PMP concentration (7.56 × 10 12} PMP / mL) and PMP particle size (70.3 nm +/- 12.4 nm (SD)) are determined by NanoFCM using the concentration and particle size standards provided by the manufacturer. bottom. The grapefruit (GF) or lemon (LM) PMP produced was used for lipid extraction using the Bright-Dyer method, as described below.

b) Modification of PMP with cationic lipids In order to prepare a lipid rearrangement PMP (LPMP), total lipid extraction from a concentrate of grapefruit or lemon PMP was performed using the Bligh-Dyer method (Bligh and). Dyer, J Biolchem Physiol, 37: 911-917, 1959). Briefly, 1 mL of concentrated PMP (10 ¹² to ¹³ PMP / mL) was mixed with 3.5 mL of a chloroform: methanol mixture (1: 2, v / v) and vortexed firmly. Next, 1.25 mL of chloroform was added, vortexed, and then stirred with 1.25 mL of sterile water. Finally, the mixture was centrifuged at 300 g for 5 minutes at room temperature. The lower organic layer containing the lipid was recovered and completely dried using the TurboVap® system (Biotage®). To modify the lipid composition of natural LPMP, synthetic cationic lipids (DOTAP, DC-cholesterol) are dissolved in chloroform: methanol (9: 1) to 25% or 40% (w / w) of the total lipid. PMP-extracted lipids were added to an amount and then vigorously mixed. The dried lipid thin film was prepared by evaporation of the solvent with an inert gas stream (eg, nitrogen) or evaporation with the TurboVap® system (FIG. 11). To prepare the reconstituted PMP from the extracted lipid, water or buffer (eg PBS) was added to the dried lipid thin film and left at room temperature for 1 hour for hydration. The formed lipid particles were subjected to 10 freezing-thaw cycles or sonication (Branson 2800 sonic bath, 10 minutes, room temperature). Next, in order to reduce the number of lipid bilayers and the overall particle size, lipid PMPs were made in 0.8 μm, 0.4 μm and 0.2 μm using Mini Extruder (Avanti® Polar Lipids). It was extruded through a polycarbonate filter (Fig. 11). If concentrated LPMP was required, the samples were concentrated by ultracentrifugation at 100,000 xg over 30 minutes at 4 ° C. The final pellet was resuspended in sterile UltraPure water or PBS and then stored at 4 ° C. until the next use. The final LPMP concentration and median LPMP particle size (89-104 nm) were determined by NanoFCM using the concentration and particle size standards provided by the manufacturer. The surface charge (zeta potential) was measured by a dynamic light scattering method using a Zetasizer (Malvern Functional). The LPMP particle size and concentration range is 83 ± 19 nm and 1.7 × 10 ¹² LPMP / mL for LM LPMP, 106 ± 25 nm and 6.54 × 10 ¹⁰ LPMP / mL for DOTAP modified LPMP, and DC-cholesterol modification. The PMP was 91 ± 17 nm and 3.08 × 10 ¹¹ LPMP / mL (Fig. 12). Modifications of the lipid rearrangement PMP with the cationic lipids DOTAP and DC cholesterol altered the surface charge of the LPMP: as the cationic lipid content increased, the surface charge of the LPMP increased (FIG. 13A). Analysis of Cryo-EM images of LPMP reconstituted from extracted lemon lipids confirmed the sphericity and particle size distribution of LPMP (68.7 ± 23 nm (SD)) (FIGS. 14A and 14B).

c) Loading on cationic lipid-modified PMPs Loading on siRNA / TracrRNA As described above, GF or LM-extracted lipids were supplemented with cationic lipids and dried for loading into siRNA / TracrRNA. SiRNA / TracrRNA dissolved in nuclease-free water or Duplex buffer (IDT®) was added to the dry lipid thin film at 1.5 nmol per 1 mg of PMP lipid and left at room temperature for 1 hour for hydration. The formed lipid particles were subjected to 10 freezing-thaw cycles and extruded through a 0.8 μm, 0.4 μm and 0.2 μm polycarbonate filter using a Mini Extruder (Avanti® Polar Lipids). (Fig. 11). Load PMPs were dialyzed against PBS overnight in a dialysis machine equipped with a 100 kDa MWCO membrane (Spectrum®) and then sterilized using a 0.2 μm polyether sulfone (PES) filter. In addition, the sample was purified and concentrated using ultracentrifugation. The loaded PMP was centrifuged at 100,000 xg for 30 minutes at 4 ° C., the supernatant was removed, the pellet was resuspended in 1 mL PBS and concentrated at 100,000 xg over 30 minutes. .. The resulting pellets were resuspended in water (for cell uptake by plant cells). The particle size and number of particles of RNA-loaded LPMP were assessed by NanoFCM: average particle size and particle concentration of 89 ± 15 nm and 1.54 × 10 ¹² LPMP / mL for unmodified LPMP, 104 for DC-Chol. For ± 25 nm and 2.54 × 10 ¹¹ LPMP / mL and for DOTAP, it was 100 ± 30 nm and 9.7 × 10 ¹¹ LPMP / mL. RNA loading was determined by Quant-iT ™ RiboGreen ™ assay or measurement of the fluorescence intensity of the labeled cargo (siRNA labeled with Alexa Fluor 555 or TracrRNA labeled with ATTO 550). The morphology and particle size of the unmodified LPMP was further analyzed by a Cryogenic electron microscope (Cryo-EM) (FIGS. 14A and 14B). The RiboGreen ™ assay was performed according to the manufacturer's protocol in the presence of heparin (5 mg / mL) and 1% Triton-X100 to lyse the PMP and release the encapsulated cargo. Modification of LPMP with cationic lipids DOTAP and DC-cholesterol altered the surface charge of LPMPs and increased loading of loaded electric cargo (eg, RNA) compared to LPMPs without cationic lipids. (FIGS. 4A-4D).

d) Increased delivery of RNA to plant cells by DC-cholesterol modified PMP Black Mexican sweet (BMS) cells from Arabidopsis Biological Resources Center (ABRC) I bought it from. BMS cells are 4.3 g / L of Murashige and Skoog base salt mixture (Sigma M5524), 2% sucrose (S0389, Millipore Sigma), 2 mg / L of 2,4-dichlorophenoxyacetic acid (D7299, Millipore Sigma), 250 μg / It was grown in Murashige and Skoog base medium (pH 5.8) containing 1 × MS vitamin solution in L thiamine HCL (V-014, Millipore Sigma) and ddH2O. This 1 x vitamin mixed solution contains niacin (N0761-100G, Millipore Sigma) and pyroxidine hydrochloride (P6280-25G) at final concentrations of 1.3 mg / L, 250 μg / L, 250 μg / L, 130 mg / L and 200 mg / L. , Millipore Sigma), D-pantothenic acid hemicalcium salt (P5155-100G, Millipore Sigma), L-asparagine (A4159-25G, Millipore Sigma) and myo-inositol (I7508-100G) containing Millipore Si. Cells were grown in a 1 L vented conical sterile flask in the dark at 24 ° C. with stirring (110 rpm).

For BMS cell treatment, 10 mL of cell suspension was harvested and the% pack cell volume (PCV) was determined. PCV is defined as the quotient of cell volume divided by the total volume of cell culture aliquots and is expressed as a percentage. The cells were centrifuged at 3900 rpm for 5 minutes to determine the volume of cell pellets. The% PCV of BMS was 20%. For uptake experiments, the% PCV of the culture was adjusted to 4% by diluting the cells with the medium as described above. LPMPs modified with LPMP and DC-cholesterol were loaded with TracrRNA labeled with ATTO 550 as described above, sterilized and resuspended in sterile water. The average particle size and concentration of the particles, analyzed by NanoFCM, were 104 ± 25 nm and 2.54 × 10 ¹¹ LPMP / mL for DC-Chol and 89 ± 15 nm and 1.54 × 10 for unmodified LPMP. ^{It was 12} LPMP / mL. The amount of TracrRNA ATTO 550 (IDT) in the sample was quantified by Quant-iT ™ RiboGreen®. Both 50 μL LPMP and DC-cholesterol modified LPMP containing 433 ng TracrRNA were added in duplicate to an aliquot of 450 μL plant cell suspension in a 24-well plate. 50 μl of ultrapure water was added to the cells and used as a negative control. The cells were incubated in the dark at 24 ° C. for 3 hours and washed 3 times with 1 mL of ultrapure water to remove particles that were not taken up by the cells. The cells were resuspended in 500 μL ultrapure water for imaging on an epifluorescence microscope (Olympus IX83). Variable fluorescence signals could be detected in plant cells treated with LPMP and LPMP modified with DC-cholesterol as compared to negative controls (ultrapure water sterile water) with no detectable fluorescence (FIG. 15). .. LPMP modified with DC-cholesterol presented the strongest fluorescent signal, indicating that this PMP modification had the strongest delivery of TracrRNA to plant cells. Our data show that modification of PMP with the cationic lipid DC-cholesterol improved uptake of lemon LPMP by plant cells in vitro.

Example 19: Modification of plants by PMP-mediated gRNA delivery This example describes the loading and functional delivery of short-stranded nucleic acids in plants to affect gene expression. This example further demonstrates that short-stranded nucleic acid load PMPs are stable and retain their activity in plants. In this example, transgenic dCas9-SunTag-VP64 Arabidopsis thaliana is used as a model plant, grapefruit PMP is used as a model PMP, and a guide RNA (gRNA) for FLOWERING WAGENINGEN (FWA) is used as a model short-stranded nucleic acid. It is used as (about 35 kDa).

a) Incorporation of lipid-reconstituted PMPs by intact plants We have previously demonstrated that natural PMPs from lemons and grapefruits can be incorporated by the roots of intact plants, including Arabidopsis thaliana. (Example 15). In this example, we demonstrate that lipid rearrangement PMP (LPMP) can also be incorporated by the roots of Arabidopsis. Lipids were extracted and reconstituted from natural lemon (LM) and grapefruit (GF) PMP as described in Example 18. The LPMP thus obtained was labeled with a near-infrared fluorescent lipophilic dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindotricarbocyanine iodide (Dir) (Thermo Fisher). .. Note that 3 μL of DiR stock solution (5 mg / mL) was added to 1 mg of PMP lipids extracted from grapefruit and lemon. The dried lipid thin film was prepared as described in Example 18, then sonicated (10 minutes, room temperature, Branson 2800 ultrasonic bath) and 0.8 μm using a Mini Extruder (Avanti Polycarbonate Lipids). Extruded through 0.4 μm and 0.2 μm polycarbonate filters. Unbound dyes were removed using a Zeba ™ rotary desalting column (40 kDa MWCO, Thermo Fisher Scientific) equilibrated with water. The Dir-labeled PMP was concentrated on an Amicon® Ultra-0.5 centrifuge filter device (Molecular Weight Cutoff (MWCO) 100 kDa).

Washing and sterile filtration of DiR-labeled LPMP The preparation is 1: 5 liquid 1/2 MS medium (2.15 g / L Murashige and Skoog base salt mixture (Sigma), 10 g / L sucrose, 0.5 g / L. It was diluted by adjusting with MES (Sigma) and KOH (pH 5.8)), and 100 μL was equally divided into a sterile 1.5 mL Eppendorf tube. Two-week-old Arabidopsis seedlings (Col-0 wild type) were transferred into prepared Eppendorf tubes using sterile forceps. Note that only the third bottom of the root is soaked. Five seedlings were used in individual tubes for each treatment (untreated control, DiR control, LM-LPMP-DiR, GF-LPMP-DiR). The tubes were sealed, covered with aluminum foil and placed in a plant growth incubator for 48 hours. The seedlings were then washed twice with water containing 1% Triton X-100 (Sigma) and once with water alone. The seedlings were towel dried and then placed on an iBright 1500 imaging system (Thermo Fisher) for imaging at 750 nm (Diro dye wavelength). Enhanced fluorescence signals were observed in plants treated with LM-LPMP-DiR and GF-LPMP-DiR compared to controls (FIGS. 9A and 9B), indicating uptake of LPMP by the plants.

b) Modification and loading of PMP by gRNA Modification and loading of PMP used in this example are described in Example 18. Natural lemon PMP was used as a lipid source, and two formulations: (1) unmodified LPMP and (2) LPMP (DC-Chol-LPMP) supplemented with DC-cholesterol were used. The guide RNA (gRNA) used in this example was obtained from Integrated DNA Technologies (IDT). An Alt-R® CRISPR-Cas9 crRNA XT (23bp) sequence targeting the Arabidopsis gene FLOWERING WAGENINGEN (FWA) (At4g25530) to form a functional gRNA double helix according to the manufacturer's instructions. Annealed to general purpose CRISPR-Cas9 tracrRNA (67bp) according to the manufacturer's instructions. Two crRNA sequences: FWA-g4 5'-ACGGAAAGATTGTATGGGGCTT-3'(SEQ ID NO: 53) and FWA-g175'-AAAAACTAGGCCATCCATTGGA-3' (Papikian et al., Nat Commun, 10: Article 29) SEQ ID NO: 54) was used. The gRNA double helices 4 and 17 (SEQ ID NOs: 53 and 54) were mixed in equimolar amounts and loaded into the LPMP as described in Example 18. The loaded LPMP preparation is sterilized (0.2 um sterile filter), washed using an ultracentrifugation approach (100,000 g, 30 minutes, 4 ° C)) and placed in 150 μL sterile water after ultracentrifugation. It was resuspended. Particle concentration and median particle size were assessed using NanoFCM and quantified RNA loading using Quant-iT ™ RiboGreen ™ RNA Assay Kit (Thermo Fisher) as described in Example 18. (Table 2).

c) Functional delivery of gRNA-loaded lemon PMP targeting FWA in dCas9-SunTag-VP64 Arabidopsis thaliana For this embodiment, we present in transgenic Arabidopsis thaliana. -The SunTag-VP64 system was used (Papikian et al., Nat Commun, 10: Article number 729, 2019). In this two-module system, deactivated Cas9 endonuclease (dCas9) is fused to the GCN4 peptide repeat to allow multiple copies of VP64 to bind to the single-stranded dCas9 protein for transcriptional activity. The conjugation factor VP64 is fused to a single-stranded variable fragment GCN4 antibody. By supplying a gRNA (FWA in this case) corresponding to the promoter sequence of the gene of interest, this dCas9-SunTag-VP64 (hereinafter referred to as SunTag) system is a "homing device" for activating gene expression. Can be used as. Transgenic seeds of the SunTag Arabidopsis thaliana plant (Papician et al., Nat Commun, 10: Article number 729, 2019) were obtained from the Arabidopsis Biological Resources Center (ABRC). The seeds were sterilized as follows: washed 3 times with sterile deionized water for 1 minute in 70% ethanol, 10 minutes in 50% household bleach with 0.1% Triton X-100 (Sigma). bottom. Sterile seeds were resuspended in sterile 0.1% agarose and stratified at 4 ° C. for 3 days in the dark. The seeds were then supplemented with 35 mg / L hygromycin B (GoldBio) to select SunTag-positive seedlings among the isolated T2 populations, 1/2 MS plate (Murashige and Skoog basic salt mixture (Sigma) 2). .15 g / L, sucrose 10 g / L, MES (Sigma) 0.5 g / L, phytoagar (Duchefa) 5 g / L, pH 5.8) adjusted with KOH. The plates were sealed with medical tape (3M) and placed in a plant growth incubator using light for 6 hours to stimulate germination. The plates were then coated with aluminum foil for 48 hours, uncoated and the seedlings were grown under normal growth conditions (16 hours light, 23 ° C / 21 ° C day / night) for 3 days. Hygromycin B-tolerant seedlings exhibited a typical darkening phenotype (extended hypocotyls compared to non-tolerant seedlings; non-tolerant seedlings died). This selection protocol was previously described in Harrison et al. , Plant Methods, 2: Article number 19, 2006.

Resistant seedlings (8 per treatment, pooled) were harvested from plates using sterile forceps and cationic or ionizable with unmodified LPMP containing 19-27 μg gRNA and 0.6 μg gRNA. It was placed in a sterile 2 mL Eppendorf tube containing 200 μL of liquid 1/2 MS medium modified with lipids and 50 μL of LPMP. The treatments were (1) LPMP-gRNA, (2) DC-Chol-LPMP-gRNA, (3) DC-Chol-LPMP-tracrRNA and (4) gRNA alone (27 μg). Care was taken to immerse the roots in the medium and leave the cotyledons floating on the surface. Eight seedlings were used per treatment. The sealed tube allows the gRNA to integrate into the dCas9-SunTag-VP64 complex in order to allow time for the PMP to be picked up by the plant roots to move through the plant and to deliver their gRNA cargo into the plant cells. Then, in order to activate the expression of the FWA gene which is not expressed in the seedling, it was placed in a plant growth incubator for 24 or 48 hours (Fujimoto et al., PLoS Genet., 4: e1000048, 2008). After the prescribed incubation time, the seedlings were removed from the PMP-containing solution, rinsed once with UltraPure water (Thermo Fisher), towel dried and frozen in liquid nitrogen.

Inverse transcriptional quantitative PCR (RT-qPCR) was used to assay mRNA transcription levels to determine activation of FWA gene expression. Frozen seedlings were ground in a tube using a plastic mortar and total RNA was extracted using RNeasy® Plant Mini Kit® according to the manufacturer's instructions. Purified RNA was eluted from the column with 40 μL of RNase-free water (Qiagen). Genomic DNA was removed from the RNA preparation using DNase I, Amplifier Grade kit (Sigma), according to the manufacturer's instructions. cDNA synthesis was performed using Invitrogen SuperScript ™ III First-Strand Synthesis Super Mix (Thermo Fisher) and Oligo (dT) primers according to the manufacturer's instructions. To detect the expression of the FWA gene (At4g25530), quantitative PCR was performed using Luna® Universal qPCR Master Mix (NEB) according to the manufacturer's instructions, Applied Biosystems ™ Quant Studio ™. It was performed on a Real-Time PCR system (Thermo Fisher). Primers used for the qPCR experiment were previously published (Cabanillas et al., Plant Cell, 30: 2594-261, 2018; Papikian et al., Nat Commun, 10: Array number 729, 2019): FWA_F. 5'-TTGGATCCAAAGGAGTATCAAAAG-3'(SEQ ID NO: 55), FWA_R 5'-CTTGGTACCAGCGGAGA-3'(SEQ ID NO: 56), IPP2_F 5'-GTATGAGTTGCTTCCAGAGAG-3'(SEQ ID NO: 57), IPP2AG SEQ ID NO: 58), Ubiquitin_F 5'-CCTACCTTTGAGGGGCTTCT-3'(SEQ ID NO: 59), Ubiquitin_R 5'-GAAGTCGTGAGAGACGCGTG-3'(SEQ ID NO: 60). qPCR was performed using the following programs: (a) 95 ° C. for 60 seconds; (b) 95 ° C. for 15 seconds and 60 ° C. for 30 seconds with 40 iterations. The ubiquitin gene (At2g36060) was used to normalize the results. As an internal control, ATISOPENTEYL DIPHOSPHE ISOMERASE 2 (IPP2) (At3g02780) was used. Activation of FWA gene expression in various treatments was determined by calculating normalized ΔΔCt values (FIGS. 10A-10C). Plants exposed to DC-cholesterol-modified LPMP loaded with equimolar amounts of gRNAs 4 and 17 for 48 hours showed a 3-fold increase in FWA gene expression compared to gRNA controls (FIG. 10B). DC-Chol LPMP containing tracrRNA control did not induce FWA expression (FIGS. 10A and 10B). No significant difference in the expression of IPP2 (housekeeping gene) was found between DC-Chol and the tracrRNA control (FIG. 10C).

Other Embodiments Some embodiments of the present invention are included in the paragraphs numbered below.
1. 1. A plant-modifying composition comprising a plurality of plant messenger packs (PMPs), wherein PMP is a plant-modifying composition comprising a plant-modifying agent.
2. 2. The plant modification composition according to paragraph 1, wherein PMP in the composition is an effective concentration for modifying plant traits, the modification increases the fitness of the plant.
3. 3. A plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP contains a plant modifier, and the PMP in the composition is a concentration effective for modifying plant traits and is modified. Is a plant modification composition that increases the adaptability of plants.
4. The plant modification composition according to paragraph 2 or 3, wherein the increase in fitness is an increase in development, proliferation, yield, resistance to abiotic stressors or resistance to biological stressors.
5. Increased plant adaptability includes disease resistance, drought resistance, heat resistance, cold resistance, salt resistance, metal resistance, herbicide resistance, drug resistance, water utilization efficiency, nitrogen utilization, nitrogen stress resistance, nitrogen fixation, pest resistance. , Herbicide resistance, pathogen resistance, yield, yield under water shortage conditions, vitality, growth, photosynthetic ability, nutrition, protein content, carbohydrate content, oil content, biomass, sprout length, root length, root structure, seed weight or harvest The plant modification composition according to paragraph 2 or 3, which is an increase in the amount of possible product.
6. The plant modification composition according to paragraph 2 or 3, wherein the increased fitness of the plant is an improvement in the quality of the product harvested from the plant.
7. The plant modification composition according to paragraph 6, wherein the increased fitness of the plant is an improvement in the taste, appearance or shelf life of the product harvested from the plant.
8. The plant modification composition according to paragraph 2 or 3, wherein the increased fitness is a reduction in the production of allergens that stimulate an immune response in the animal.
9. The plant-modifying composition according to any one of paragraphs 1 to 8, wherein the plant-modifying agent is a heterologous nucleic acid.
10. A plant modification composition comprising multiple plant messenger packs (PMPs), the PMP containing a heterologous nucleic acid, and the PMP in the composition being an effective concentration for increasing the fitness of the plant. Modified composition.
11. The plant modification composition according to paragraph 9 or 10, wherein the heterologous nucleic acid is a DNA, RNA, PNA or hybrid DNA-RNA molecule.
12. The plant modification composition according to paragraph 11, wherein the RNA is messenger RNA (mRNA), guide RNA (gRNA) or inhibitory RNA.
13. The plant modification composition according to paragraph 12, wherein the inhibitory RNA is RNAi, shRNA or miRNA.
14. The plant modification composition according to paragraph 12 or 13, wherein the inhibitory RNA inhibits the expression of a gene in a plant.
15. Nucleic acids are mRNAs, modifications that increase the expression of enzymes, pore-forming proteins, signaling ligands, cell membrane permeabilizing peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins, riboproteins, protein aptamers or chaperons in plants. The plant modification composition according to paragraph 9 or 10, which is an mRNA or DNA molecule.
16. Nucleic acids include antisense RNAs, siRNAs, shRNAs, miRNAs that reduce the expression of enzymes, transcription factors, secretory proteins, structural factors, riboproteins, protein aptamers, chapelons, receptors, signaling ligands or transport factors in plants. The plant modification composition according to paragraph 9 or 10, which is an aiRNA, PNA, morpholino, LNA, piRNA, ribozyme, DNAzyme, aptamer, cyclRNA, gRNA or DNA molecule.
17. The plant modification composition according to paragraph 9 or 10, wherein the heterologous nucleic acid is a non-integrated construct that expresses the nucleic acid in a plant.
18. The plant modification composition according to paragraph 9 or 10, wherein the heterologous nucleic acid is an integrated construct that expresses the nucleic acid in a plant.
19. The plant-modifying composition according to any one of paragraphs 1 to 8, wherein the plant-modifying agent is a heterologous peptide.
20. A plant modification composition comprising multiple plant messenger packs (PMPs), wherein the PMP comprises a heterologous peptide, and the PMP in the composition is a concentration effective for increasing the fitness of the plant. Modified composition.
21. The plant modification according to paragraph 19 or 20, wherein the peptide is an enzyme, a pore-forming protein, a signaling ligand, a cell membrane penetrating peptide, a transcription factor, a receptor, an antibody, a Nanobody, a gene editing protein, a riboprotein, a protein aptamer or a chaperone. Composition.
22. The plant modification composition according to paragraph 19 or 20, wherein the peptide reduces gene expression in a plant.
23. The plant modification composition according to paragraph 19 or 20, wherein the peptide increases the expression of a gene in a plant.
24. The plant modification composition according to any one of paragraphs 1 to 23, wherein PMP comprises two or more various plant modifiers.
25.2 The plant modification composition according to paragraph 24, wherein the two or more various plant modifiers contain heterologous nucleic acids and heterologous peptides.
26. The plant modifying composition according to any one of paragraphs 1 to 25, wherein the plant modifying agent is encapsulated by PMP.
27. The plant modification composition according to any one of paragraphs 1 to 25, wherein the plant modification agent is embedded on the surface of PMP.
28. The plant modification composition according to any one of paragraphs 1 to 25, wherein the plant modification agent is conjugated to the surface of PMP.
29. The plant modifying composition according to any one of paragraphs 1 to 28, wherein the plant modifying agent is non-insecticidal.
30. The plant modification composition according to any one of paragraphs 1 to 29, wherein the composition is stable at room temperature for at least 1 day and / or at 4 ° C. for at least 1 week.
31. The plant modification composition according to any one of paragraphs 1-29, wherein PMP is stable for at least 24 hours, 48 hours, 7 days or 30 days.
32. The plant modification composition according to paragraph 31, wherein PMP is stable at a temperature of at least 4 ° C, 20 ° C, 24 ° C or 37 ° C.
33. The plant modification composition according to any one of paragraphs 1-22, wherein the PMP in the composition is a concentration of at least 1, 10, 50, 100 or 250 μg / ml of PMP protein.
34. PMP is the plant modification composition according to any one of paragraphs 1-3, comprising purified extracellular vesicles (EV) or fragments or extracts thereof.
35. The plant modification composition according to paragraph 34, wherein the plant EV is a modified plant extracellular vesicle (EV).
36. The plant modification composition according to any one of paragraphs 1 to 35, wherein the plant is an agricultural plant or a horticultural plant.
37. The plant modification composition according to paragraph 36, wherein the agricultural plant is a soybean plant, a wheat plant or a corn plant.
38. The plant modification composition according to any one of paragraphs 1-37, wherein the composition is formulated for delivery to a plant.
39. The plant modification composition according to paragraph 38, wherein the composition is formulated for delivery to a plant leaf, seed, root, fruit, sprout or flower.
40. The plant modification composition according to any one of paragraphs 1 to 39, wherein the composition comprises an agriculturally acceptable carrier.
41. The plant modification composition according to any one of paragraphs 1 to 40, which is formulated to stabilize PMP.
42. The plant modification composition according to any one of paragraphs 1-41, which is formulated as a liquid, solid, aerosol, paste, gel or gas composition.
43. A pest control composition comprising a plurality of PMPs, wherein the PMP is (a) a step of providing an initial sample from the plant or a portion thereof, wherein the plant or a portion thereof comprises EV; (b). In the step of isolating the crude PMP fraction from the initial sample, the crude PMP fraction is at least one contaminant or unwanted from the plant or portion thereof at a reduced level relative to the level in the initial sample. Steps with components; (c) Purification of the crude PMP fraction, thereby producing multiple pure PMPs, wherein the multiple pure PMPs are reduced relative to the levels in the crude EV fraction. Steps; (d) loading the plant modification composition into multiple pure PMPs; and (e) to the plant, having at least one contaminant or unwanted component from the plant or parts thereof at the indicated levels. A plant modification composition produced by a process comprising the step of formulating the PMP of step (d) for delivery.
44. A plant comprising the plant modification composition according to any one of paragraphs 1-43.
45. A method of delivering a plant modification composition to a plant, comprising contacting the plant with the composition according to any one of paragraphs 1-43.
46. A method for increasing the fitness of a plant, comprising delivering to the plant an effective amount of the plant modification composition according to any one of paragraphs 1-43, as compared to untreated plants. How to increase the fitness of plants.
47. 45. The method of paragraph 45 or 46, wherein the plant modification composition is delivered to a leaf, seed, root, fruit, sprout, flower or portion thereof.
48. A method of increasing the fitness of a plant, comprising contacting the plant with an effective amount of the plant modification composition according to any one of paragraphs 1-43, comprising contacting the plant as compared to an untreated plant. How to increase fitness.
49. A method for increasing the fitness of a plant, comprising contacting the meristem of the plant with an effective amount of the plant modification composition according to any one of paragraphs 1-43, the untreated plant. How to increase the fitness of plants compared to.
50. A method for increasing the fitness of a plant, comprising contacting the embryo of the plant with an effective amount of the plant modification composition according to any one of paragraphs 1-43, comprising contacting the untreated plant. How to increase the fitness of plants in comparison.
51. A method of increasing the fitness of a plant, comprising contacting the protoplast of the plant with an effective amount of the plant modification composition according to any one of paragraphs 1-43, as compared to untreated plants. How to increase the fitness of plants.
52. A method of increasing the adaptability of a plant, comprising contacting the plant cells of the plant with an effective amount of the plant modification composition according to any one of paragraphs 1-43, as compared to untreated plants. And how to increase the adaptability of plants.
53. The method of any one of paragraphs 46-52, wherein the increase in fitness is an increase in development, proliferation, yield, resistance to abiotic stressors or resistance to biological stressors.
54. Increased plant adaptability includes disease resistance, drought resistance, heat resistance, cold resistance, salt resistance, metal resistance, herbicide resistance, drug resistance, water utilization efficiency, nitrogen utilization, nitrogen stress resistance, nitrogen fixation, pest resistance. , Herbicide resistance, pathogen resistance, yield, yield under water shortage conditions, vitality, growth, photosynthetic ability, nutrition, protein content, carbohydrate content, oil content, biomass, sprout length, root length, root structure, seed weight or The method of paragraphs 46-52, which is an increase in the amount of product that can be harvested.
55. The method of paragraphs 46-52, wherein increasing the fitness of the plant is an improvement in the quality of the product harvested from the plant.
56. The method of paragraph 55, wherein the improvement in quality is an improvement in the taste, appearance or shelf life of the product harvested from the plant.
57. The method of paragraphs 46-52, wherein increasing fitness is a reduction in the production of allergens that stimulate an immune response in an animal.
58. The method according to any one of paragraphs 45-57, wherein the plant is an agricultural plant or a horticultural plant.
59. 58. The method of paragraph 58, wherein the plant is a soybean plant, a wheat plant or a corn plant.
60. The method of any one of paragraphs 45-59, wherein the plant modification composition is delivered as a liquid, solid, aerosol, paste, gel or gas.
61. The plant modification composition according to any one of paragraphs 1-43, wherein PMP is a lipid rearrangement PMP (LPMP).
62. LPMP is the plant modification composition according to paragraph 61, which comprises an exogenous cationic lipid.
63. Each of the modified PMPs comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% cationic lipids. The plant modification composition according to paragraph 61 or 62.
64. The plant modification composition according to any one of paragraphs 61 to 63, wherein the cationic lipid is DC-cholesterol or DOTAP.
65. A method of administering a heterologous RNA to a plant, comprising delivering a composition comprising multiple plant messenger packs (PMPs) to the plant, each comprising the heterologous RNA, wherein the composition is a plant adaptation. A method of delivery at an effective concentration to increase the degree.
66. The method according to paragraph 65, wherein the heterologous RNA is a guide RNA (gRNA).
67. The method of paragraph 66, wherein the gRNA is a component of the ribonuclear protein complex (RNP), where each of the plurality of PMPs comprises an RNP.
68. 65. The method of paragraph 65, wherein PMP is a lipid rearrangement PMP (LPMP).
69. The method of paragraph 65, wherein the heterologous RNA is encapsulated by PMP.
70. 65. The method of paragraph 65, wherein each of the PMPs comprises purified plant extracellular vesicles (EVs).
71. 65. The method of paragraph 65, wherein PMP in the composition is an effective concentration for modifying plant traits, the modification increasing the fitness of the plant.
72. 65. The method of paragraph 65, wherein the increase in plant fitness is an increase in development, proliferation, yield, resistance to abiotic stressors or resistance to biological stressors.
73. The method of paragraph 65, wherein the increased fitness of the plant is a modification of the flowering time.
74. The method of paragraph 65, wherein increasing the fitness of the plant is an improvement in the quality of the product harvested from the plant.
75. 65. The method of paragraph 65, wherein increasing the fitness of the plant is an improvement in the taste, appearance or shelf life of the product harvested from the plant.
76. 65. The method of paragraph 65, wherein increasing fitness is a reduction in the production of allergens that stimulate an immune response in an animal.

The invention described above has been described in some detail as description and examples for clarity, but such descriptions and examples should not be construed as limiting the scope of the invention. The disclosure of all patents and scientific literature cited herein is expressly incorporated by reference in its entirety.

Other embodiments are included in the claims.

Claims (17)

A method of administering a heterologous RNA to a plant comprising delivering a composition comprising a plurality of plant messenger packs (PMPs) to the plant, each comprising the heterologous RNA, wherein the composition comprises the heterologous RNA. , A method, delivered at an effective concentration to increase the adaptability of the plant. The method according to claim 1, wherein the heterologous RNA is a guide RNA (gRNA). The method according to claim 2, wherein the gRNA is a component of a ribonucleoprotein complex (RNP), and each of the plurality of PMPs comprises the RNP. The method of claim 1, wherein the PMP is a liquid reconstituted PMP (LPMP). The method of claim 1, wherein the heterologous RNA is encapsulated by the PMP. The method of claim 1, wherein each of the PMPs comprises a purified plant extracellular vesicle (EV). The method of claim 1, wherein the PMP in the composition is an effective concentration for modifying the traits of the plant, the modification increasing the fitness of the plant. The method of claim 1, wherein the increase in fitness of a plant is an increase in development, proliferation, yield, resistance to abiotic stressors or resistance to biological stressors. The method of claim 1, wherein the increase in fitness of the plant is a modification of the flowering time. The method of claim 1, wherein the increase in fitness of the plant is an improvement in the quality of the product harvested from the plant. The method of claim 1, wherein the increase in fitness of the plant is an improvement in the taste, appearance or shelf life of the product harvested from the plant. The method of claim 1, wherein the increase in fitness is a reduction in the production of allergens that stimulate an immune response in an animal. A plant-modifying composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP comprises a plant-modifying agent. A plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP contains a plant modifier, and the PMP in the composition is at a concentration effective for modifying plant traits. Yes, the modification is a plant modification composition that increases the adaptability of the plant. A plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP comprises a heterologous nucleic acid, and the PMP in the composition is at an effective concentration for increasing the fitness of the plant. There is a plant modification composition. A plant modification composition comprising a plurality of plant messenger packs (PMPs), wherein the PMP comprises a heterologous peptide, and the PMP in the composition is at a concentration effective for increasing the fitness of the plant. There is a plant modification composition. A plant modification composition containing a plurality of PMPs, wherein the PMPs are
(A) A step of providing an initial sample from a plant or portion thereof, wherein the plant or portion thereof comprises an EV;
(B) In the step of isolating the crude PMP fraction from the initial sample, the crude PMP fraction is at least from the plant or portion thereof at a reduced level relative to the level in the initial sample. Steps with one contaminant or unwanted component;
(C) The step of purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs are at reduced levels relative to the levels in the crude EV fraction. With at least one contaminant or unwanted component from said plant or parts thereof;
(D) A plant modification produced by a process comprising loading the plant modifier into the plurality of pure PMPs; and (e) formulating the PMP in step (d) for delivery to the plant. Composition.

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